WO2016042411A1 - Peptides ncx1 et leurs utilisations - Google Patents

Peptides ncx1 et leurs utilisations Download PDF

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Publication number
WO2016042411A1
WO2016042411A1 PCT/IB2015/002029 IB2015002029W WO2016042411A1 WO 2016042411 A1 WO2016042411 A1 WO 2016042411A1 IB 2015002029 W IB2015002029 W IB 2015002029W WO 2016042411 A1 WO2016042411 A1 WO 2016042411A1
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peptide
ncxl
seq
plm
peptidomimetic
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PCT/IB2015/002029
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English (en)
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Pimthanya WANICHAWAN
Ole M. SEJERSTED
Cathrine Rein Carlson
Kjetil HODNE
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University Of Oslo
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Heart failure often referred to as chronic heart failure (CHF) occurs when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs.
  • CHF chronic heart failure
  • Heart failure Common causes of heart failure include coronary artery disease including a previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, valvular heart disease, excess alcohol use, infection, and cardiomyopathy of an unknown cause. These cause heart failure by changing either the structure or the functioning of the heart.
  • the drug that has been used for over 250 years to treat heart failure, digitalis, is still in use, but with very limited indications today. That is because the drug does not improve survival and carries the risk of cardiac arrhythmias and sudden death.
  • Digitalis acts by inhibiting the Na,K-pump. This causes intracellular Na to rise and thereby reduce the driving force for the NCX1 to pump Ca out of the cell (see Aronsen et al, J Mol Cell Cardiol 2013, 61 : 11-19 for a review). Hence, the cardiac cells may become overloaded with Ca which will improve contractility, but also give rise to triggered arrhythmias.
  • the present invention relates to peptides and their therapeutic and research use.
  • the present invention provides NCXl -PLM disruption peptides and methods of using such peptides as heart disease therapeutic agents.
  • peptides are conjugated to membrane translocating peptides (e.g., TAT or variants thereof), or with other molecules known to faciliate intracellular uptake. Such conjugation may be either at the C or N terminal ends or internally.
  • the peptides are chemically modified by various means to improve stability.
  • the peptides are expressed intracellularly from exogenously delivered nucleic acid constructs.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the peptides (e.g., the peptides of SEQ ID NOs: 11-56, and 59-74 and peptides that are at least 85% identical to SEQ ID Nos 11-56, and 59-74 (e.g., at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NOs: 1 1-56, and 59-74).
  • the composition increases NCXl activity in a cell (e.g., a cardiomyocyte).
  • the composition treats or prevents heart disease or related conditions (e.g., chronic heart disease, chronic heart failure, congestive heart failure, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, cardiomyopathy with systolic and/or diastolic dysfunction, and cardiac hyperthrophy).
  • heart disease or related conditions e.g., chronic heart disease, chronic heart failure, congestive heart failure, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, cardiomyopathy with systolic and/or diastolic dysfunction, and cardiac hyperthrophy.
  • Further embodiments provide a method or use of increasing NCXl activity in a cell, comprising administering any of the aforementioned peptides to a cell.
  • Additional embodiments provide a method or use of treating or preventing heart disease, comprising administering any of the aforementioned peptides to a subject in need thereof.
  • FIG. 1 shows analyses of the PLM-NCX1 interaction.
  • A Rat left ventricle lysate was subjected to immunoprecipitation using anti-NCXl.
  • B Schematic presentation of biotinylated peptides covering PLM cyt and pSer68-PLM cyt (upper panel). Immunoblot analysis of the two biotinylated peptides using anti-biotin-HRP is shown in lower panel.
  • FIG. 2 shows identification of pSer68-PLM and PLM binding sites down to the amino acid level in the cytoplasmic part of NCXl.
  • A Biotin-PLM or
  • B Biotin-pSer68-PLM binding was identified by overlaying the two peptides on membranes containing 20-mer overlapping NCXl peptides and immunoblotting with anti-biotin-HRP (upper panels).
  • C Schematic illustration of four biotinylated NCXl peptides used in D.
  • D Pull-down assay with four biotinylated NCXl peptides against PLM expressed in HEK293 cells. PLM binding was analyzed by immunoblotting with anti-PLM. Beads were used as a negative control (upper panel). Bar graph show relative PLM binding semi quantified by densitometry analysis.
  • FIG. 3 shows optimization of pSer68-PLM binding sequences.
  • NCXl 333-352 (KHPDKEIEQLIELANYQVLS, SEQ ID NO: 4) was synthesized as a two dimensional peptide array and overlayed with biotin-pSer68-PLM.
  • FIG. 4 shows that the optimized peptide has increased affinity for PLM.
  • biotin-NCXl (333-352) (native sequence), biotin-NCXl (K333Y, D336Y) (optimized sequence) and biotin-NCXl -scrambled (control sequence) peptides used in pull down assay in B.
  • B Pull-down assays with biotin-NCXl (333-352) and biotin-NCXl (K333Y, D336Y) against PLM (S68D) expressed in HEK293 cells.
  • FIG. 5 shows that the optimized peptide reverses PLM (S68D) inhibition of NCXl.
  • FIG. 6 shows a model of PLM (S68D) inhibition of NCXl activity and relieve of its inhibition by the optimized peptide.
  • pSer68-PLM phosphorylation denoted with a yellow star
  • Opt-Pep an optimized anchoring disruptor peptide, derived from pSer68-PLM binding sequences in NCXl, relieves the inhibitory effect of pSer68-PLM, but does not cause the total dissociation of Ser68-PLM from NCXl.
  • FIG. 7 shows two-dimensional peptide array of NCXl (324-343).
  • NCXl (324- 343) (ARILKELKQKHPDKEIEQLI, SEQ ID NO: 3) was synthesized as a two dimensional peptide array and overlayed with biotin-pSer68-PLM.
  • FIG. 8 shows analyses of the relative biotin-pSer68-PLM binding to various NCXl (333-352) derivatives.
  • A K5Y-,
  • B P3Y-,
  • C H2Y-,
  • D I7Y- and
  • E IlY-containing NCXl (333-352) sequences.
  • the presented data is an average from two independent peptide arrays.
  • FIG. 9 shows systematic truncations of a leading peptide (Opt-pep).
  • Opt-pep was also systematic substituted with glycine (G) at either C- or N-terminus (right panels in A and B, respectively).
  • G glycine
  • the biotin-pSer68-PLM binding was detected using anti-biotin-HRP.
  • the minimum Opt-pep sequence for pSer68-PLM binding is PYKEIEQLIELANYQV (SEQ ID NO: 73) (miniOpt-pep, 16 amino acids) as indicated in C.
  • FIG. 10 shows analyses of miniOpt-pep substitutions and biotarget response.
  • B Ten Opt-pep derived sequences that were synthesized in solution.
  • C Schematic presentation of the two PLM constructs used in this study: PLM-2xHis6 and PLM-3xFLAG.
  • F The pSer68-PLM level in rat left ventricle lysate, pre-incubated with or without Optl-Opt6, was analyzed by immunoblotting using a specific pSer68-PLM antibody.
  • FIG. 1 1 shows whole cell patch clamp analysis of NCXl regulation by Opt-pep on adult ventricular cardiomyocytes.
  • the isolated Ni 2+ sensitive NCXl currents plotted in a current (I)- voltage (V) curve with the current (ordinate) normalized to cell capacitance (pA/pF) and -voltage on the abscissa in mV.
  • FIG. 12 shows In vivo of Opt-pep efficacy in mice.
  • A Biotin-pSer68-PLM overlay on the native NCXl sequence (333-352), Opt-pep, PR-Opt-pep and scrambled peptide control synthesized on membrane.
  • B safety of Opt-pep treatment in the normal heart
  • C efficacy studies of Opt-pep in disease model (mice with diastolic dysfunction).
  • D Immunoblotting of the Opt-pep peptide separated on SDS-PAGE gel using a specific Opt-pep antibody.
  • E Immunoblotting of Opt-pep expressed in HEK293 cells using a specific Opt-pep antibody.
  • the term "subject” refers to organisms to be treated by the methods of embodiments of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.
  • the term “subject” generally refers to an individual who will receive or who has received treatment (e.g., administration of a peptide of the present invention and optionally one or more other agents) for heart disease or other condition requiring treatment.
  • diagnosis refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, diagnostic assay (e.g., for heart failure) and the like.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments include, but are not limited to, test tubes and cell cultures.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • the term "host cell” refers to any eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • eukaryotic or prokaryotic cell e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells
  • the term "cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • the term "effective amount” refers to the amount of a therapeutic agent (e.g., a peptide of the present invention) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • co-administration refers to the administration of at least two agent(s) (e.g., a peptide of the present invention) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In some embodiments, a first agent/therapy is administered prior to a second agent/therapy.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are coadministered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g. , toxic) agent(s).
  • the term "toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975]).
  • sample as used herein is used in its broadest sense.
  • a sample may comprise a cell, tissue, or fluids, nucleic acids or polypeptides isolated from a cell, and the like.
  • the terms “purified” or “to purify” refer, to the removal of undesired components from a sample.
  • substantially purified refers to molecules that are at least 60% free, preferably 75% free, and most preferably 90%, or more, free from other components with which they usually associated.
  • Amino acid sequence and terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • native protein as used herein to indicate that a protein does not contain amino acid residues encoded by vector sequences; that is, the native protein contains only those amino acids found in the protein as it occurs in nature.
  • a native protein may be produced by recombinant means or may be isolated from a naturally occurring source.
  • portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein.
  • the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample.
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • test compounds are agents that treat or prevent heart failure.
  • the present invention relates to peptides and their therapeutic and research use.
  • the present invention provides NCX1-PLM disruption peptides and methods of using such peptides as heart disease therapeutic agents.
  • the Na + -Ca 2+ exchanger (NCX 1) is one of the essential regulators of Ca
  • NCX1 plays a critical role in maintaining the balance of Ca 2+ flux across the sarcolemmal membrane in excitation-contraction coupling (Bers, Nature 2002, 415: 198-205). Cardiac muscle contracts in response to the rise in [Ca 2+ ]i which is released from the sarcoplasmic reticulum (SR) and from in flux across the sarcolemma through voltage-sensitive channels.
  • SR sarcoplasmic reticulum
  • SR Ca 2+ - ATPase recycles Ca 2+ from the cytosol into the lumen of the SR, and NCX1 mediates the movement of [Ca 2+ ]i across the sarcolemma to the extracellular space.
  • the exchanger catalyzes the electrogenic exchange of Ca 2+ and Na + across the plasma membrane and can work in both directions, either in the Ca 2+ -in flux (reverse) or Ca 2+ -efflux (forward) mode.
  • NCXl transports approximately 28% of the cytosolic Ca 2+ during a contraction- relaxation cycle in large animals and humans, with 70% being reaccumulated in the SR (via SERCA) (Bassani and Bers, J Mol Cell Cardiol 1994, 26: 1335-1347; Bers and Bridge, Circ Res 1989, 65:334-342; Bers et al, Am J Physiol 1990, 258:C944-954). Alterations in any of the activities associated with this complex process cause a corresponding change in the amount of Ca 2+ released from the SR and the resulting force of cardiac contraction.
  • NCXl is electrogenic since it carries three Na ions in exchange for one Ca. Since the Na,K-pump keeps intracellular Na under control, the NCX is driven by the membrane potential and the Na-gradient. Therefore, in a resting cell these two parameters will determine the intracellular Ca 2+ concentration ([Ca 2+ ]i). Generally, variation in expression level of NCXl has much smaller effect on [Ca 2+ ]i in the resting cell than changes in membrane potential or intracellular Na + .
  • Phospholemman is a transmembrane protein in the heart and a substrate for protein kinase A and protein kinase C. Phosphorylation of PLM at serine 68 (pSer68-PLM) results in inhibition of NCXl activity. pSer68-PLM level is upregulated in heart failure in rabbit (Bossuyt et al, Circ Res 2005, 97:558-565).
  • the drug that has been used for over 250 years to treat heart failure, digitalis, is still in use, but with very limited indications today. That is because the drug does not improve survival and carries the risk of cardiac arrhythmias and sudden death.
  • Digitalis acts by inhibiting the Na,K-pump. This causes intracellular Na to rise and thereby reduce the driving force for the NCXl to pump Ca out of the cell (see Aronsen et al, J Mol Cell Cardiol 2013, 61 : 11-19 for a review). Hence, the cardiac cells may become overloaded with Ca which will improve contractility, but also give rise to triggered arrhythmias.
  • NCX 1 primarily works in forward mode, it is reversed early during the action potential bringing Ca 2+ into the cell. By doing this it contributes to triggering and synchronizing Ca 2+ release from the sarcoplasmic reticulum. Thus, relieving the inhibition of NCX in heart failure causes more rapid rise of Ca 2+ so that slow contractions become faster and systolic function is improved. During diastole the NCX operates in forward mode again, carrying Ca 2+ out of the cell. By speeding up this process, diastolic Ca 2+ is reduced especially at high heart rates. Diastolic dysfunction, i.e.
  • peptide anchoring disrupters for the functional NCXl-pSer68- PLM interaction. Strong binding of pSer68-PLM to NCX1-PASKT and NCX1-QKHPD regions was demonstrated in pull-down and overlay assays. Two-dimensional peptide arrays of NCX1-PASKT and NCX1-QKHPD revealed that the NCX1-QKHPD region (1- KHPDKEIEQLIELANYQVLS-20, SED ID NO: 11) was a candidate to optimize as amino acid substitutions within the sequence increased pSer68-PLM binding.
  • substitutions include, but are not limited to, K1C, K1F, K1W, K1Y, H2R, H2Y, P3F, P3R, P3W, P3Y, D4X (any amino acid), K5C, K5F, K5L, K5W, K5Y, E6X (any amino acid), I7W, ⁇ 7 ⁇ , E8C, E8M, E8R, E8S, Q9I, Q9L, Q9V, Q9Y, L10I, L10V, L10Y, and 111Y (SED ID NOs 12-42).
  • Combinations of individually preferred substitutions were evaluated, leading in many cases to double-substituted peptides with improved activity.
  • Particularly preferred combinations include, but are not limited to, those involving K1Y or K5Y, including, K1Y/D4Y, K1Y/D4W, K1Y/D4F, K1Y/D4V, K1Y/E6Y, K1Y/E6F, K1Y/E6Q, K1Y/E6V, K1Y/E6S, K1Y/E6W, K5Y/D4W, K5Y/D4F, K5Y/D4Y, K5Y/D4V (SEQ ID NOs 43-56).
  • K1Y/D4Y One of the preferred double-substituted 20-mers, K1Y/D4Y (Oct-pep, SED ID NO 43) was chosen to illustrate the potential for reducing the size of the peptides while retaining functionality.
  • Selected particularly strong binding peptides include Optl (PFKEIEQLIELANYQV, SEQ ID NO 65), Opt 2 (PYHEIEQLIELANYQV, SEQ ID NO: 66) Opt 3 (PYMEIEQLIELANYQV, SEQ ID NO: 67) , Opt4 (PYREIEQLIELANYQV, SEQD ID NO: 68) and Opt 6 (PYKEYEQLIELANYQV, SEQ ID NO: 70). Additionally, Oct8 (PYKEIEQLIELKNYQV, SEQ ID NO: 72) and Oct 9 (PYKEIEQLIELRNYQV, SEQ ID NO: 73) increased PLM oligomerization and represent useful therapeutic agents.
  • NCX1-PLM protein- protein interaction offers a new therapeutic approach for heart disease.
  • NCX1 -derived peptides 16-20 amino acid length NCX1 -derived peptides.
  • the invention is not restricted to peptides of this length, and it is envisioned that interacting peptides of any size derived from or comprising the described sequence could be useful, administered either in chemically synthesized form, or expressed from a suitable nucleic acid vector (e.g., SEQ ID NOs: 59-74).
  • the core interacting peptide sequence may be modified moderately, or conjugated with other structures, in a manner retaining the interacting properties, in order to facilitate intracellular uptake or provide increased serum stability or improved pharmacokinetic properties.
  • Embodiments of the present invention provide peptides that inhibit the functional interaction of NCX1 and PLM, variants (e.g., variants that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such peptides), mimetics, or modified versions thereof.
  • the peptides correspond to the interaction domain of NCX1 (e.g., amino acids 333-352) with PLM, although other regions are specifically contemplated.
  • peptides are between 5 and 30 amino acids (e.g., 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, etc.). Exemplary peptides are described in SEQ ID NOs: 1 1-56, and 59-74.
  • Percent sequence identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).
  • 1 , 2, 3, or 4 amino acids from the peptides described herein may be deleted. In some embodiments, 1, 2, 3, or 4 amino acids may be inserted into the peptides or added to either the C or N terminal end. In some embodiments, 1 , 2, 3, or 4 amino acids within the peptides may be replaced with other amino acids. Suitable amino acid substitutions include conservative amino acid substitutions.
  • individual amino acid substitutions can be selected from any one of the following: 1) the set of amino acids with nonpolar sidechains, for example, Ala, Cys, He, Leu, Met, Phe, Pro, Val; 2) the set of amino acids with negatively charged side chains, for example, Asp, Glu; 3) the set of amino acids with positively charged sidechains, for example, Arg, His, Lys; and 4) the set of amino acids with uncharged polar sidechains, for example, Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, to which are added Cys, Gly, Met and Phe.
  • the set of amino acids with nonpolar sidechains for example, Ala, Cys, He, Leu, Met, Phe, Pro, Val
  • the set of amino acids with negatively charged side chains for example, Asp, Glu
  • 3) the set of amino acids with positively charged sidechains for example, Arg, His, Lys
  • a naturally occurring amino acid can also be replaced with, for example, a non-natural amino acid such as, for example, norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al, Meth. Enzym., 1991, 202, 301-336.
  • a non-natural amino acid such as, for example, norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al, Meth. Enzym., 1991, 202, 301-336.
  • the procedures of Noren et al., Science, 1989, 244, 182 and Ellman et al, supra can be used.
  • the peptides described herein can further be modified. Some modifications may increase the stability and activity of a peptide to enable reduced dosing level or frequency, as well as enable alternative routes of administration, e.g., oral or inhalation.
  • the following are examples of modifications of peptides that may increase stability, activity, specificity, and/or efficacy.
  • D-amino acids are unnatural amino acids which are less likely to be attacked by proteases.
  • a protease cleavage site prediction program has identified 8 cleavage sites for trypsin.
  • one or more L-arginines (R) in the peptide can be replaced with D-arginines as described by Powell et al. (Pharm. Res., 1993, 10, 1268-1273.)
  • Oligomerize the peptide Oligomerize the peptide.
  • Cyclizing a peptide may protect it against proteolysis and degradation. Cyclizing the peptide head to tail can be accomplished by linking the amino terminus to the carboxy terminus as described by Marastoni et al. (Arzneiffenaba, 1994, 44, 1073-1076). In addition, cyclizing a peptide may occur via side-chain to side-chain. Further, cyclizing a peptide may occur through commonly used coupling methods using agents such as, for example, p-nitrophenyl esters, the azide method, 2,4,5-trichlorophenyl and pentafluorophenyl esters and the mixed anhydride method.
  • 1-benzotriazole-tris-dimethyl aminophosphonium hexafluorophosphate BOP
  • O- (benzotriazol-l-yl)-l, 1, 3, 3-tetramethyl uronium hexafluorophosphate HBTU
  • 1- benzotriazolyloxy-tris-pyrrolidino-phosphonium hexafluorophosphate PyBOP
  • O- (benzotriazol-l-yl)-l, 1,3, 3-tetramethyl uronium tetrafluoroborate TBTU
  • 7-azabenzotriazol- 1-yloxytrispyrrolidino phosphonium hexafluorophosphate PyAOP
  • HATU 7-azabenzotriazol-l-yloxy- tris-dimethyl aminophosphonium
  • Biodegradable modifications e.g., polymers of N-acetylneuraminic adid (poysialic acids) as described in, for example, Georgiadis et al. (Cell. Mol. Life Sci., 2000, 57, 1964- 1969).
  • Carriers such as liposomes, microspheres or microcapsules, poly lactic acid (PLA), poly lactic/glycolic acid
  • PLGA PLGA as described in, for example, Heya et al. (J. Pharm. Sci., 1994, 83, 798-801), nanoparticles and emulsions, cyclodextrins and derivatives.
  • Formulations that protect peptides such as those containing different types of protease inhibitors.
  • formulation containing multifunctional polymers which exhibit mucoadhesive properties as well as enzyme inhibitory activity e.g., poly(acrylates), thiolated polymers, and polymer-enzyme inhibitor conjugates.
  • Examples include, but are not limited to, hydrophilic (e.g., Penetratin or Antenapedia PTD RQIKWFQNRRMKWKK (SEQ ID NO: 76); TAT YGRKKRRQRRR (SEQ ID NO: 77); SynBl RGGRLS YSRRRF ST STGR (SEQ ID NO: 78); SynB3 RRLSYSRRRF (SEQ ID NO: 79); PTD-4 PIRRRKKLRRLK (SEQ ID NO: 80); PTD-5 RRQRRT SKLMKR (SEQ ID NO: 81); FHV Coat-(35-49)
  • hydrophilic e.g., Penetratin or Antenapedia PTD RQIKWFQNRRMKWKK (SEQ ID NO: 76); TAT YGRKKRRQRRR (SEQ ID NO: 77); SynBl RGGRLS YSRRRF ST STGR (SEQ ID NO: 78); SynB3 RRLSYSRRRF (SEQ
  • RRRRNRTRRNRRRVR (SEQ ID NO: 82); BMV Gag-(7-25)
  • KMTRAQRRAAARRNRWTAR (SEQ ID NO: 83); HTLV-II Rex-(4-16)
  • TRRQRTRRARRNR SEQ ID NO: 84
  • D-Tat GRKKRRQRRRPPQ SEQ ID NO: 85
  • R9- Tat GRRRRRRRRRPPQ SEQ ID NO: 86
  • amphiphilic e.g., transportan
  • GALFLGWLGAAGSTMGAWSQPKKKRKV SEQ ID NO: 90
  • MPG ac- GALFLGFLGAAGSTMGAWSQPKKKRKV-cya SEQ ID NO: 91
  • MPG(ANLS) ac- GALFLGFLGAAGSTMGAWSQPKSKRKV-cya SEQ ID NO: 92
  • Pep- 1 ac- KETWWETWWTEWSQPKKKRKV-cya SEQ ID NO: 93
  • Pep-2 ac- KETWFETWFTEWSQPKKKRKV-cya SEQ ID NO: 94)
  • peptidomimetics of the peptides described herein are provided.
  • the use of peptides as lead compounds, and subsequently conversion into low-molecular- weight nonpeptide molecules (peptidomimetics), have successfully led to development of small-molecule antagonists of intracellular targets (Bottger et al, J. Mol. Biol, 1997, 269, 744-56; Bottger et al, Oncogene, 1996, 13, 2141-7). Therefore, peptidomimetics have emerged as a powerful means for overcoming the obstacles inherent in the physical characteristics of peptides, improving their therapeutic potential (Kieber-Emmons et al, Curr. Opin.
  • peptidomimetics possess desirable pharmacodynamic properties superior to natural peptides, including good oral activity, long duration of action, better transport through cellular membranes, decreased rate of excretion, and decreased hydrolysis by peptidases.
  • peptide ligands such identified further serve as starting points for a combinatorial chemistry approach or a medicinal chemistry-based peptidomimetic approach for the development of new directed therapeutic agents.
  • the determination of the structural basis for the high-binding affinity of these peptides for their substrate contributes to the rational design of a therapeutic agent.
  • the present disclosure also provides nucleic acid molecules encoding any of the peptides disclosed herein, vectors comprising any of the nucleic acid molecules disclosed herein, compositions comprising any of the nucleic acid molecules or vectors disclosed herein, as well as host cells comprising any of the nucleic acid molecules or vectors disclosed herein.
  • peptides are synthesized de novo.
  • a variety of peptide synthesis methods may be utilzed. Examples include, but are not limited to, solid-phase peptide synthesis (SPPS), (R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149-2154; Mitchell, A. R. K., S.B.H.; Engelhard, M.; Merrifield, R.B. (1978). "A new synthetic route to tert- butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin, an improved support for solid-phase peptide synthesis". J.
  • SPPS The general principle of SPPS is one of repeated cycles of coupling-wash- deprotection-wash.
  • the free N-terminal amine of a solid-phase attached peptide is coupled (see below) to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
  • the superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.
  • SPPS Fmoc and Boc.
  • solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by either of these two groups and added onto a deprotected amino acid chain.
  • the present invention further provides the use and methods of using the peptides described herein in the disruption of NCX1-PLM interaction.
  • Such peptides find use in the activation of NCX1 in a cell (e.g., a cardiomyocyte) and in the treatment of heart disease and related conditions.
  • diseases and conditions treated by the peptides described herein include but are not limited to chronic heart disease; chronic heart failure, congestive heart failure, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, cardiomyopathy with systolic and/or diastolic dysfunction, and cardiac hypertrophy.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • peptides described herein are introduced either subcutaneous ly or intravenously.
  • peptides are administered using a gene delivery technique to express the peptide in taget (e.g., cardiac) cells.
  • taget e.g., cardiac
  • recombinant adeno-associated virus (serotype 9) vectors carrying peptide-encoding cDNA with a cytomegalovirus promoter (AAV9) (Cutler MJ, et al. Circulation. 2012; 126:2095- 2104; herein incorporated by reference in its entirety) are utilized.
  • AAV9 cytomegalovirus promoter
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.
  • Thickeners flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the active agents of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the active agents of the formulation.
  • Dosing is dependent on severity and responsiveness of the disease state or condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • the administering physician can readily determine optimum dosages, dosing methodologies and repetition rates.
  • Example 1 is provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
  • Example 1 is provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
  • Peptides on cellulose membranes were synthesized using Multipep automated peptide synthesizer (I TAVIS Bioanalytical Instruments AG, Koeln, Germany) (Frank, R. (1992) Tetrahedron 48, 9217-9232). Parts of the intracellular loop of rat CXl (EDM02743) was synthesized as overlapping 20-mer peptides with three amino acid offsets. Peptides in solution were synthesized and purified to > 80% purity by Genscript (Corporation,
  • Biotin-NCXl (261-280): Biotin-KRYRAGKQRGMIIEHEGDRP (SEQ ID NO: 1) Biotin-NCXl (267-286): Biotin-KQRGMIIEHEGDRPASKTEI (SEQ ID NO: 2)
  • Biotin-NCXl (324-343): B iotin- ARILKELKQKHPDKEIEQLI (SEQ ID NO: 3)
  • Biotin-NCXl 333-352: Biotin-KHPDKEIEQLIELANYQVLS (SEQ ID NO: 4)
  • Biotin-PLM (58-92): Biotin-KRCRCKFNQQQRTGEPDEEEGTFRSSIRRLSSRRR (SEQ ID NO: 5)
  • Biotin-pSer68-PLM (58-92): KRCRCKFNQQQRTGEPDEEEGTFRSSIRRLpSSRRR (SEQ ID NO: 6)
  • Biotin-NCXl (333 -352): Biotin-KHPDKEIEQLIELANYQVLS (SEQ ID NO: 7)
  • NCX1 ⁇ 333 -352, I7Y NCX1 ⁇ 333 -352, I7Y: KHPDKEYEQLIELANYQVLS (SEQ ID NO: 30)
  • NCX1 ⁇ 333 -352, E8M KHPDKEIMQLIELANYQVLS (SEQ ID NO: 32)
  • NCX1 ⁇ 333 -352, Q9L KHPDKEIELLIELANYQVLS (SEQ ID NO: 36)
  • NCX1 ⁇ 333 -352, L10V NCX1 ⁇ 333 -352, L10V: KHPDKEIEQVIELANYQVLS (SEQ ID NO: 40)
  • NCX1 ⁇ 333 -352, L10Y NCX1 ⁇ 333 -352, L10Y: KHPDKEIEQYIELANYQVLS (SEQ ID NO: 41)
  • NCX1 ⁇ 333 -352, K1Y/D4W YHPWKEIEQLIELANYQVLS (SEQ ID NO: 44)
  • NCX1 ⁇ 333 -352, K1Y/D4V NCX1 ⁇ 333 -352, K1Y/D4V: YHPVKEIEQLIELANYQVLS (SEQ ID NO: 46)
  • NCX1 ⁇ 333 -352, K1Y/E6Y YHPDKYIEQLIELANYQVLS (SEQ ID NO: 47)
  • NCX1 ⁇ 333 -352, K1Y/E6F NCX1 ⁇ 333 -352, K1Y/E6F: YHPDKFIEQLIELANYQVLS (SEQ ID NO: 48)
  • NCX1 ⁇ 333 -352, K1Y/E6Q YHPDKYIEQLIELANYQVLS (SEQ ID NO: 49)
  • NCX1 ⁇ 333 -352, K1Y/E6V NCX1 ⁇ 333 -352, K1Y/E6V: YHPDKYIEQLIELANYQVLS (SEQ ID NO: 50)
  • NCX1 ⁇ 333 -352, K1Y/E6S YHPDKSIEQLIELANYQVLS (SEQ ID NO: 51)
  • NCX1 ⁇ 333 -352, K1Y/E6W NCX1 ⁇ 333 -352, K1Y/E6W: YHPDKWIEQLIELANYQVLS (SEQ ID NO: 52)
  • NCX1 ⁇ 333 -352, K5Y/D4Y KHPYYEIEQLIELANYQVLS (SEQ ID NO: 55)
  • NCXl(333-352, K5Y/D4V) KHPVYEIEQLIELANYQVLS (SEQ ID NO: 56)
  • HEK293 cells were cultured in DMEM (Gibco-BRL) supplemented with 10% fetal calf serum (Gibco-BRL), 100 unit/ml of penicillin, 0.1 mg/ml of streptomycin
  • Plasmid DNA was transfected into HEK293 cells using Lipofectamine 2000 as instructed by the manufacturer (Invitrogen). After 24 hours, the cells were lysed in lysis buffer containing 20 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA and 0.5% Triton X-100 with complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany).
  • Frozen LV from rats were pulverized in a mortar with liquid nitrogen followed by adding ice-cold lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 % Triton) supplemented with 1 mM PMSF (93482, Sigma-Aldrich) and a Complete Mini EDTA-free tablet (Roche Diagnostics, Mannheim, Germany).
  • Tissue samples were homogenized three times for 1 min on ice with a Polytron 1200 and centrifuged at 100 000 g for 60 min at 4°C. Supernatants were collected and stored at -70°C.
  • Synthesized peptide arrays (SPOT) membranes were first activated by soaking membranes in methanol for few seconds and followed by three washes with TBS-T (tris- buffered saline with 0.1% tween). The membranes were then incubated with blocking solution (1% casein) (Roche Diagnostics) at room temperature. After one hour blocking, the membranes were incubated with 1 ⁇ biotinylated peptide in blocking solution overnight at 4°C with gentle agitation. The membranes were then washed three times with TBS-T (3 x l0min). Binding was detected by immunoblotting. Pull-down assay with biotinylated peptides
  • the mixture of peptide-bound beads and protein samples was rotated for 2 hours at 4°C followed by washing the beads three times with IP buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) prior to boiling in 2X SDS loading buffer. Binding was analysed by immunoblotting.
  • Immunoprecipitation was performed by incubating 2 ⁇ g of the appropriate antibody with protein samples (rat heart lysates) and protein A/G agarose beads (sc-2003, Santa Cruz Biotechnology) overnight at 4°C. Next day, the samples were then washed three times in IP buffer and boiled in SDS loading buffer before SDS-PAGE analysis. Equal amount of rabbit IgG (sc-2027) was used as negative control. Blocking peptide (antigen: cardiac NCX1, sequence: CGQPVFRKVHARDHPIPST) (Genscript) was incubated with anti-NCXl prior immunoprecipitation (negative control).
  • Anti-NCXl epiplasmic NCX1 protein with as N-terminal HIS tag and Trigger Factor (TF) to stabilize the protein (HIS-TF-NCX1) was custom made from Genscript Corp.
  • the cells were clamped to -43 mV (reversal potential) for 6 min to allow sufficient intracellular diffusion of both ions and peptides.
  • the NCX current was recorded after a pre pulse protocol and immediate from -43 mV, a descending voltage ramp from 120 mV to -100 mV was elicited.
  • the NCX current was isolated using 5 ⁇ Ni 2+ added in the EC. The currents were normalized to the cell capacitance and the current (I) - voltage (V) relations were plotted from -100 to 100 mV.
  • Densitometric analysis was performed using Image Gauge V4.0, ImageQuant TL (Amersham Biosciences) or Image J (USA).
  • biotin-PLM cyt and biotin-pSer68-PLM cy t were overlayed overlapping 20-mer peptides covering the PLM binding part of NCXl.
  • Both peptides bound to PASKT- and QKHPDK-containing sequences in NCXl Fig. 2A-B, marked in bold letters
  • both peptides bound to the self-dimerization XIP domain in NCXl Fig.2A-B, underscored sequence).
  • peptides can be synthetized with a different yield, depending on the peptide sequence (hydrophobicity and confirmation) (Katz, C, Levy-Beladev, L., Rotem- Bamberger, S., Rito, T., Rudiger, S. G., and Friedler, A. (2011) Chemical Society reviews 40, 2131-2145), the four NCXl peptides covering amino acids 261-280, 267-286, 324-343, and 333-352 (black arrows in Fig. 2A-B) were also synthesized as purified biotinylated peptides in solution (Fig. 2C) and analysed for binding to exogenous PLM expressed in HEK293.
  • the two-dimensional peptide arrays of the two NCX1-KHPDK containing sequences given in Fig. 2A-B were further optimized by two dimensional peptide arrays where each residue in the native peptide sequences were systemically substituted with every possible residue.
  • Substitution of NCXl 333-352 (Fig. 3A, KHPDKEIEQLIELANYQVLS (SEQ ID NO: 4)
  • NCXl 324-343
  • Fig.7A ARILKELKQKHPDKEIEQLI (SEQ ID NO: 3)
  • Binding affinity was dramatically increased when the NCXl (333-352) peptide was substituted in position 1-1 1 (labelled with red circles) compared to internal control peptides (white circles). Few substitutions were accepted for lysine in position 1 (Kl) and histidine in position 2 (H2), whereas aspartate in position 4 (D4) and glutamate in position 6 (E6) were highly flexible (denoted with arrows above the array). Tryptophan (W) and tyrosine (Y) substitutions highly increased the affinity for pSer68-PLM (strong signals).
  • NCXl (333-352) peptide was single substituted with a tyrosine (Y) in position Kl, H2, P3, K5, 17 and 111.
  • K1Y, H2Y, P3Y, and K5 greatly increased the affinity for
  • Biotin-pSer68-PLM (Fig. 2B).
  • each of these single mutations was combined with various amino acid substitutions in position D4 and E6.
  • Kl Y, D4Y which showed a somewhat increased affinity for pSer68-PLM
  • other double mutations had a lower pSer68- PLM affinity compared to that of Kl Y (Fig. 8A-E).
  • the optimized peptide increases stabilization of the PLM-NCX1 complex in left ventricle lysate
  • the currents were recorded in whole cell patch clamp configuration and acquired using ramp protocol from 120 to -100 mV.
  • the Ni 2+ sensitive currents, corresponding to the isolated NCX1 current, were plotted as an I-V curve from -100 to 100 mV.
  • the assays for measuring the effect of PLM (S68D) where first validated without adding the optimized peptide.
  • the normal NCX1 shown in Fig. 5 (circle) had a robust current in both forward and reverse mode. Consistent with literature, co-transfecting NCX1 with PLM (S68D) reduced the currents significantly (triangle). Adding a scramble peptide did not relive the inhibition of phosphorylated PLM (diamond). On the other hand, adding the optimized peptide relived the PLM inhibition (inverted triangle) and the current returned to within the range seen with only NCX1 transfected cells.
  • Peptides on cellulose membranes were synthesized using Multipep automated peptide synthesizer (INT AVIS Bioanalytical Instruments AG, Koeln, Germany). Peptides in solution were synthesized and purified to obtain > 80% purity by Genscript (Corporation, Piscataway, NJ) (listed below). A biotin tag was included at the N-terminus for one peptide.
  • Biotin-pSer68-PLM KRCRCKFNQQQRTGEPDEEEGTFRSSIRRLpSSRRR (SEQ ID NO: 6)
  • PR-Opt-pep PR- YHPYKEIEQLIELANYQVLS (SEQ ID NO: 57)
  • Opt-pep-dellC YHPYKEIEQLIELANYQVL (SEQ ID NO: 59)
  • Opt-pep-del2C YHPYKEIEQLIELANYQV (SEQ ID NO: 60)
  • Opt-pep-dellN HPYKEIEQLIELANYQVLS (SEQ ID NO: 61)
  • Opt-pep-del2N PYKEIEQLIELANYQVLS (SEQ ID NO: 62)
  • Opt-pep-dellNIC HPYKEIEQLIELANYQVL (SEQ ID NO: 63)
  • Peptide membranes were first activated by soaking in methanol for few seconds. The activated membrane was thereafter washed three times with TBS-T (Tris-buffered saline with 0.1% tween) and incubated with blocking solution (1% casein) (Roche Diagnostics, Mannheim, Germany) at room temperature. After one hour blocking, the membranes were incubated with 5 ⁇ Biotin-pSer68-PLM peptide in 1% casein overnight at 4°C with gentle agitation. Next day, washing of the membranes were performed three times 10 minutes in TBS-T. Binding was visualized by immunoblotting.
  • TBS-T Tris-buffered saline with 0.1% tween
  • blocking solution 1% casein
  • the membranes were incubated with 5 ⁇ Biotin-pSer68-PLM peptide in 1% casein overnight at 4°C with gentle agitation. Next day, washing of the membranes were performed three times 10 minutes in TBS-T. Bin
  • HEK293 cells were cultured in DMEM (Gibco-BRL) supplemented with 10% fetal calf serum (Gibco-BRL), 100 unit/ml of penicillin, 0.1 mg/ml of streptomycin
  • Plasmid DNA was transfected into HEK293 cells using the CaC . method. After 24 hours, the cells were lysed in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.5% Triton X-100 with complete protease inhibitor cocktail tablets (Complete Mini EDTA-free, Roche Diagnostics, Mannheim, Germany).
  • Immunoprecipitation was performed by incubating 2 ⁇ g of the appropriate antibody with HEK293 lysates and protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4°C with rotation. The next day, samples were washed three times in IP buffer and boiled in 2 x SDS loading buffer prior to SDS-PAGE analysis. I mm un oh lotting.
  • Anti-biotin-HRP (AO 185, 1 :2000) and anti-FLAG (F1804, 1 :2000) was purchased from Sigma-Aldrich (St. Louis, MO).
  • Anti-His (A00186-100, 1 : 1000) was purchased from Genscript (Corporation, Piscataway, NJ).
  • Anti-Opt-pep epitope
  • the patch electrodes were made from borosilicate glass with filament, and the electrode resistance was 1-2 ⁇ .
  • the patch pipettes were filled with a solution comprising in mM; 125 Cs-Asp, 5 MgATP, 5 Na 2 ATP, 1 MgCl, 10 EGTA, 10 HEPES, 6 CaCl, adjusted to 7.2 using CsOH.
  • the free Ca 2+ were during these conditions 318 nM measured using MAXchelator.
  • 5 ⁇ Opt-pep was added to the pipette solution prior to the experiments. Data were acquired at a sampling rate of 20 kHz with a 5 kHz filtration using an Axopatch 200B amplifier (Axon Instruments).
  • the NCXl reversal potential under these conditions was -20 mV at 37 °C.
  • Cells were voltage clamped at -20 mV for 5 min to allow sufficient intracellular dialysis.
  • NCXl current was elicited by a descending voltage ramp from 90 mV to -100 mV (0.05 V/s) and isolated using 5 ⁇ Ni 2+ .
  • the currents were normalized to cell capacitance and the current (I)-voltage (V) relations were plotted from -100 to 60 mV. Isolation of adult cardiomyocytes.
  • Rats were anesthetized in a chamber filled with 95% room air and 5% isoflurane (Abbott Scandinavia Ab, Solna, Sweden), and were subsequently sacrificed by cervical dislocation. Hearts were then quickly excised and placed in ice cold 0.15 M NaCl solution with heparin (Heparin LEO, 5000 IE/mL; Orifarm AS, Norway).
  • the LV was minced and gently shaken at 37°C for ⁇ - ⁇ min in the same solution used in the perfusion, but with addition of 1% Bovine Serum Albumin (BSA) (A9647, Sigma-Aldrich Corporation) and 0.02 units/ml deoxyribonuclease I (LS002006, Worthington Biochemical Corp).
  • BSA Bovine Serum Albumin
  • the digested ventricular tissue was then filtered (200 ⁇ nylon mesh) and
  • cardiomyocytes sedimented.
  • the cardiomyocyte pellet was resuspended in cell isolation buffer with 1% BSA (A9647, Sigma-Aldrich Corporation) and 0. ImM Ca 2+ in the solution. Isolated cardiomyocytes were kept at room temperature until use.
  • biotin-pSer68-PLM was overlaid C- (Fig. 9A) and N-terminally truncations (Fig. 9B) of Opt-pep (YHPYKEIEQLIELANYQVLS (SEQ ID NO: 43)), and in combination (Fig. 9C), synthesized on membranes. Binding was detected by using anti-biotin-HRP antibody. The results revealed that PYKEIEQLIELANYQV (SEQ ID NO: 64) was the minimum pSer68-PLM binding sequence (Fig. 9C). The 16 amino acids sequence was named miniOpt-pep.
  • Opt-pep is introduced into one week old mice by adeno- associated virus (AAV) technology.
  • AAV-cTNTp-RFP-2A-GOI-WPRE vector where GOI is either Opt-pep (YHPYKEIEQLIELANYQVLS-stop; SEQ ID NO:43) or a scrambled peptide sequence thereof (VYEKNYLLLPAIEQEQSHIY-stop; SEQ ID NO: 58), are custom made and packaged into AAV serotype 9 (custom AAV service by Vector Biolabs,
  • Adeno-associated virus serotype 9-mediated gene expression from the cardiac specific promotor troponin T (cTnT) is reported to be 640-fold greater in the heart compared to the next highest tissue (liver) (Prasad et al. 2011 Gene Ther. 18, 43-52).
  • Red fluorescence protein (RFP) is used as it is compatible with excitation at 488 niii (used for Fluo-4 Ca 2+ sensors and Ca 2+ imaging).
  • the short 2A peptide sequence when cloned in- frame between RFP and Opt-pep/scrambled control, allow for efficient, stoichiometric production (1 : 1) of discrete protein-peptide products within the single vector, through a novel "cleavage" event within the "self-cleavage” 2A peptide sequence.
  • the first 19 amino acids of T2A plus several linker amino acids goes to the C- terminal of RFP.
  • PR-Opt-pep PRYHPYKEIEQLIELANYQVLS-stop; SEQ ID NO: 57
  • PRVYEKNYLLLPAIEQEQSHIY-stop SEQ ID NO: 75
  • PR-Opt-pep and Opt-pep bind equally well to pSer68-PLM (Fig. 11A).
  • Fig. 11A One week old C57BL/6 mice (Jackson laboratories) have mediastinum injected with 5* 10 13 vg (vector genomes )/kg.
  • Hearts from the mice are harvested at different time points and expression of RFP/GOI is analyzed by immunohistochemistry, immunoblotting (anti- Opt-pep or anti-RFP, e.g. using ab34771 from Abeam) or RT-qPCR. Detectable expression is expected around 2 weeks. Animal characteristics are in the beginning measured every week and thereafter every second week. The safety experiment is scheduled in Fig. 1 IB. When safety of the Opt-pep treatment for the normal heart has been established, hypertrophy is induced by aortic banding at week 9. Animal characteristics are measured for the non-treated and treated animals and heart will be harvested for molecular analyses. The disease model experiment is scheduled in Fig. 1 1C.

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Abstract

La présente invention concerne des peptides, leur utilisation dans la thérapie et la recherche. La présente invention concerne également des peptides de rupture de NCX1-PLM, ainsi que des procédés d'utilisation de tels peptides comme agents thérapeutiques destinés aux maladies cardiaques.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108939075A (zh) * 2018-07-26 2018-12-07 陕西师范大学 Ncx1基因作为治疗呼吸暂停综合症的药物干预靶点的应用
US10995119B2 (en) * 2016-01-06 2021-05-04 Okinawa Institute Of Science And Technology School Corporation Peptide exhibiting hydrolytic activity and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112118A1 (en) * 1999-12-02 2005-05-26 Myriad Genetics, Incorporated Compositions and methods for treating inflammatory disorders
EP2639240A2 (fr) * 2012-02-21 2013-09-18 Consiglio Nazionale Delle Ricerche Peptides capables de neutraliser l'activité d'inhibition d'une hétérodimère MDM2/MDM4 vers p53 et leur utilisation pour le traitement du cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112118A1 (en) * 1999-12-02 2005-05-26 Myriad Genetics, Incorporated Compositions and methods for treating inflammatory disorders
EP2639240A2 (fr) * 2012-02-21 2013-09-18 Consiglio Nazionale Delle Ricerche Peptides capables de neutraliser l'activité d'inhibition d'une hétérodimère MDM2/MDM4 vers p53 et leur utilisation pour le traitement du cancer

Non-Patent Citations (59)

* Cited by examiner, † Cited by third party
Title
ARONSEN ET AL., J MOL CELL CARDIOL, vol. 61, 2013, pages 11 - 19
BASSANI; BERS, J MOL CELL CARDIOL, vol. 26, 1994, pages 1335 - 1347
BEELEY, TRENDS BIOTECHNOL., vol. 12, 1994, pages 213 - 216
BERS ET AL., AM J PHYSIOL, vol. 258, 1990, pages C944 - 954
BERS, NATURE, vol. 415, 2002, pages 198 - 205
BERS; BRIDGE, CIRC RES, vol. 65, 1989, pages 334 - 342
BIRD ET AL., METHODS ENZYMOL, vol. 446, 2008, pages 369 - 386
BIRD ET AL., PNAS, vol. 107, 2010, pages 14093 - 14098
BOSSUYT ET AL., CIRC RES, vol. 97, 2005, pages 558 - 565
BOTTGER ET AL., J. MOL. BIOL., vol. 269, 1997, pages 744 - 756
BOTTGER ET AL., ONCOGENE, vol. 13, 1996, pages 2141 - 2147
BRINCKERHOFF ET AL., INT'L J. CANCER, vol. 83, 1999, pages 326 - 334
CHANDRUDU ET AL., MOLECULES, vol. 18, 2013, pages 4373 - 4388
CHEUNG JOSEPH Y ET AL: "Coordinated Regulation of Cardiac Na+/Ca2+ Exchanger and Na+-K+-ATPase by Phospholemman (FXYD1)", SODIUM CALCIUM EXCHANGE: A GROWING SPECTRUM OF PATHOPHYSIOLOGICAL IMPLICATIONS SPRINGER, 233 SPRING STREET, NEW YORK, NY 10013, UNITED STATES SERIES : ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY (ISSN 0065-2598(PRINT)), 2013, & 6TH INTERNATIONAL CONFERENCE ON SODIUM CALCIUM EXCHANGE; LACCO AMENO, ITALY; OCTOBER 01 -05, 2011, pages 175 - 190, XP009188494 *
CUTLER ET AL., CIRCULATION, vol. 126, 2012, pages 2095 - 2104
CUTLER MJ ET AL., CIRCULATION, vol. 126, 2012, pages 2095 - 2104
CWIRLA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 6378 - 6382
DASGUPTA ET AL., BIOL. PHARMA. BULL., vol. 25, 2002, pages 29 - 36
DAVIES, J. PEPTIDE SCIENCE, vol. 9, 2003, pages 471 - 501
DEVLIN ET AL., SCIENCE, vol. 249, 1990, pages 404 - 406
DOWER, CURR. OPIN. CHEM. BIOL., vol. 2, 1998, pages 328 - 334
ELLMAN ET AL., METH. ENZYM., vol. 202, 1991, pages 301 - 336
FRANK, R., TETRAHEDRON, vol. 48, 1992, pages 9217 - 9232
GENTILUCCI ET AL., CURR PHARM DES, vol. 16, 2010, pages 3195 - 3203
GEORGIADIS ET AL., CELL. MOL. LIFE SCI., vol. 57, 2000, pages 1964 - 1969
GREEN ET AL., J. ENDOCRINOL., vol. 180, 2004, pages 379 - 388
HARRIS, GUT, vol. 35, no. 3, 1994, pages 1 - 4
HEYA ET AL., J. PHARM. SCI., vol. 83, 1994, pages 798 - 801
HODGSON; SANDERSON, CHEM SOC REV, vol. 33, 2004, pages 422 - 430
HODNE KJETIL ET AL: "Liberation of Pser68-Plm Inhibition of NCX1 by an Optimized Anchoring Disruptor Peptide", BIOPHYSICAL JOURNAL, vol. 108, no. 2, 27 January 2015 (2015-01-27), XP029193855, ISSN: 0006-3495, DOI: 10.1016/J.BPJ.2014.11.2372 *
HOJO, CURR OPIN STRUCT BIOL, vol. 26C, 2014, pages 16 - 23
J. WANG ET AL: "Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na+/Ca2+ Exchanger", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 42, 20 October 2006 (2006-10-20), US, pages 32004 - 32014, XP055249302, ISSN: 0021-9258, DOI: 10.1074/jbc.M606876200 *
JACOBSEN ET AL., J ORG CHEM, vol. 76, 2011, pages 1228 - 1238
JUFANG WANG ET AL: "Regulation of in vivo cardiac contractility by phospholemman: role of Na ϩ /Ca 2ϩ exchange", 30 December 2010 (2010-12-30), XP055249180, Retrieved from the Internet <URL:http://ajpheart.physiology.org/content/300/3/H859.full.pdf> [retrieved on 20160211] *
KATZ, C.; LEVY-BELADEV, L.; ROTEM-BAMBERGER, S.; RITO, T.; RUDIGER, S. G.; FRIEDLER, A, CHEMICAL SOCIETY REVIEWS, vol. 40, 2011, pages 2131 - 2145
KIEBER; EMMONS ET AL., CURR. OPIN. BIOTECHNOL., vol. 8, 1997, pages 435 - 441
KIM ET AL., ORG LETT, vol. 12, 2010, pages 3046 - 3049
KREBS ET AL., CHEMISTRY, vol. 10, 2004, pages 544 - 553
MADDEN ET AL., CHEM COMMUN, vol. 37, 2009, pages 5588 - 5590
MARASTONI ET AL., ARZNEIMITTELFORSCHUNG, vol. 44, 1994, pages 1073 - 1076
MARTIN: "Remington's Pharmaceutical Sciences", 1975, MACK PUBL. CO.
MILLETTI F: "Cell-penetrating peptides: classes, origin, and current landscape", DRUG DISCOV TODAY, vol. 17, 2012, pages 850 - 860
MITCHELL, A. R. K., S.B.H.; ENGELHARD, M.; MERRIFIELD, R.B: "A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin, an improved support for solid-phase peptide synthesis", J. ORG. CHEM., vol. 43, no. 13, 1978, pages 2845 - 2852
MOORE ET AL., TRENDS PHARMACOL. SCI., vol. 15, 1994, pages 124 - 129
NOREN ET AL., SCIENCE, vol. 244, 1989, pages 182
POWELL ET AL., PHARM. RES., vol. 10, 1993, pages 1268 - 1273
PRASAD ET AL., GENE THER, vol. 18, 2011, pages 43 - 52
R. B. MERRIFIELD: "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide", J. AM. CHEM. SOC., vol. 85, no. 14, 1963, pages 2149 - 2154
RAMAKERS ET AL., CHEM SOC REV, vol. 43, 2014, pages 2743 - 2756
RAMON ET AL., PHARM. RES., vol. 22, 2005, pages 1374 - 1386
SCOTT ET AL., SCIENCE, vol. 249, 1990, pages 386 - 390
SIDHU ET AL., METHODS ENZYMOL., vol. 328, 2000, pages 333 - 363
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482 - 489
STALMANS, SOFIE; WYNENDAELE, EVELIEN; BRACKE, NATHALIE; GEVAERT, BERT; D'HONDT, MATTHIAS; PEREMANS, KATHELIJNE; BURVENICH, CHRISTI: "Chemical-Functional Diversity in Cell-Penetrating Peptides", PLOS ONE, vol. 8, no. 8, pages E71752
SULOCHANA ET AL., CURR. PHARM. DES., vol. 13, 2007, pages 2074 - 2086
VERDINE; HILINSKI, METHODS ENZYMOL, vol. 503, 2012, pages 3 - 33
WANG ET AL., AM J PHYSIOL HEART CIRC PHYSIOL, vol. 300, 2011, pages H859 - 868
WHITE ET AL., METHODS, vol. 60, 2013, pages 70 - 74
XUE-QIAN ZHANG ET AL: "Residues 248 -252 and 300 -304 of the cardiac Na ϩ /Ca 2ϩ exchanger are involved in its regulation by phospholemman", AM J PHYSIOL CELL PHYSIOL, 1 January 2011 (2011-01-01), pages 833 - 840, XP055249284, Retrieved from the Internet <URL:http://ajpcell.physiology.org/content/301/4/C833.full.pdf> *

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US10995119B2 (en) * 2016-01-06 2021-05-04 Okinawa Institute Of Science And Technology School Corporation Peptide exhibiting hydrolytic activity and use thereof
CN108939075A (zh) * 2018-07-26 2018-12-07 陕西师范大学 Ncx1基因作为治疗呼吸暂停综合症的药物干预靶点的应用

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