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WO2025050091A1 - Therapeutic peptides to manage atrial fibrillation - Google Patents

Therapeutic peptides to manage atrial fibrillation Download PDF

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Publication number
WO2025050091A1
WO2025050091A1 PCT/US2024/044936 US2024044936W WO2025050091A1 WO 2025050091 A1 WO2025050091 A1 WO 2025050091A1 US 2024044936 W US2024044936 W US 2024044936W WO 2025050091 A1 WO2025050091 A1 WO 2025050091A1
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seq
composition according
peptide
greater similarity
amino acid
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PCT/US2024/044936
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French (fr)
Inventor
Mona EL REFAEY
Nipun MALHOTRA
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Ohio State Innovation Foundation
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Publication of WO2025050091A1 publication Critical patent/WO2025050091A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • Atrial fibrillation is the most common sustained clinical arrhythmia with a lifetime incidence of up to 37% and is a major contributor to morbidity and mortality. AF increases risk of heart failure, myocardial infarction, chronic kidney disease, stroke, and death. Notably, it has been over 10 years since the introduction of any new therapeutic agent for AF. AF drug development has been diminished by many factors including limited efficacy and the risk of side effects. AF causes both electrical and structural remodeling of the atria. Both processes contribute to the gradual progression of AF and further increase the duration of the AF episode and to reduce the efficacy of antiarrhythmic drugs. Thus, there is the crucial need for development of anti-arrhythmic therapies that are atrial specific.
  • K + channels such as the acetylcholine-activated potassium channel ( K A ch), formed of G- protein-gated inwardly rectifying potassium subunits (GIRK1 and GIRK4) and predominantly expressed in the atria to avoid ventricular pro-arrhythmic side effects.
  • K A ch acetylcholine-activated potassium channel
  • GIRK1 and GIRK4 G- protein-gated inwardly rectifying potassium subunits
  • GIRK4 inactivation has been proposed to target AF.
  • Aberrant / K A ch regulation is well documented and a major target for AF therapy.
  • a ch can abrogate AF.
  • tertiapin Q was shown to block other K + channels and other GIRK hetero-tetramers in addition to the GIRK1/GIRK4.
  • Other l K , A ch blockers have also been evaluated in previous studies but the field still lacks a targeted strategy. [0005] There remains a need for improved compositions and methods for the treatment of AF.
  • Figure 1 depicts how normal atrial myocytes, l-GIRK4(blue)/GIRKl(red) assemble in the ER/trans-Golgi and then is delivered to the membrane. 2- and 3- In AF, there is less GIRK1 and increased formation of GIRK4 homo-tetramers at the membrane.
  • Figure 2 depicts a schematic presentation of predicted mode of action of GIRK1 C- terminus (408-439) peptide.
  • the peptide disrupts the overall availability of free GIRK4 for GIRK4 homo-tetramer assembly by occupying the C-terminus in GIRK4 subunit.
  • GIRK4 blue
  • GIRK1 red
  • GIRK4p.G387R black circle.
  • FIG. 3 depicts the structure of modeled GIRK1/4 hetero-tetramer (Top Left).
  • the GIRK1 region (408-439, red) makes critical contact with 375-399 region in GIRK4 (Bottom Left).
  • Interaction between GIRK4 and GIRK1 is mediated by a H-bond between the side chain of lysine 426 in GIRK1 C-terminus and tyrosine 379 in GIRK4 C-terminus (Bottom Right).
  • GIRK1 and GIRK4 models were generated using l-Tasser online tool and structural figures were made using Chimera.
  • Figure 4 depicts a comparison of channel pore size among GIRK4 homo-tetramer, GIRK1 homo-tetramer and GIRK1/4 hetero-tetramer.
  • GIRK1 homo-tetramer showed significant constriction in pore opening on its intracellular side vs. extracellular side, thereby making it difficult for ions to pass. Pore opening towards extracellular and intracellular side is depicted in black and yellow circles, respectively.
  • FIG. 5 depicts that GIRK subunits bind with Flotil lin-1 (FLOT-1) and expression of GIRK1, GIRK4 and FLOT-1 in HL-1 cells.
  • FLOT-1 Flotil lin-1
  • Co-immunoprecipitation experiments were performed from mouse atrial lysates using (A) beads conjugated to GIRKl-lg or control-lg and (B) beads conjugated to GIRK4-lg or control-lg. Bound protein was immunoblotted with FLOT-1.
  • C-E Expression of GIRK4 (C), GIRK1 (D) and FLOT-1 (E) in HL-1 cells.
  • Figure 6 depicts aberrant expression of GIRK4 in dogs with persistent AF post chronic atrial pacing.
  • Figure 7 depicts a GIRK4p.G387R +/+ mouse model.
  • A-B conserveed sequence of GIRK4 binding motif across species.
  • C Representative chromatograms of the GIRK4p.G387R +/+ mice carrying the point mutation at amino acid 387 and control littermates.
  • D Kcnj5 relative expression in GIRK4p.G387R +/+ mice.
  • FIG. 9 depicts GIRK1 and GIRK4 localization at the atrial myocytes.
  • A-B Expression of GIRK1 at the membrane of atrial myocytes in WT (A, 20X; B, 63X mag).
  • C-D GIRK4 is uniformly localized at membrane of atrial myocytes (Scale bars equal 10pm).
  • E GIRK1 is uniformly expressed at the membrane of atrial myocytes in WT cells.
  • F Lost/discontinuous expression of GIRK1 at the membrane of GIRK4p.G387R +/+ atrial myocyte. Scale bars equal 20pm.
  • FIG 11 depicts that GIRK4p.G387R +/+ mice are more prone to atrial arrhythmias. Electrocardiogram representatives from programmed electric stimulation acquired from GIRK4p.G387R +/+ mice and their control littermates.
  • FIG. 12 depicts GIRK4p.G387R +/+ mice display heart rate variability.
  • FIG. 13 depicts that GIRK4p.G387R +/+ mice demonstrate a structural atrial phenotype.
  • FIG 14 depicts that GIRK4p.G387R +/+ atrial myocytes display aberrant l K ,ACh fast desensitization.
  • Figure 15 depicts the characterization of hiPSC-CMs.
  • Figure 16 depicts the electrical characterization of hiPSC-aCMs.
  • A patch clamp recordings of action potentials (APs) from hiPSC-AMs ⁇ 10 pM CCh).
  • CCh shortened APD.
  • B Increased expression of GIRK1 and GIRK4 in hiPSC-aCMs vs. hiPSC-vCMs.
  • Figure 17 depicts the effects of peptide-based drug
  • D-E Immunoblotting following surface biotinylation of HL-1 cells treated with PBS (control), peptide 1 (TAT-Gl-C-terminus) and peptide 2 (CPC-C-Gl-C-terminus).
  • Figure 18 depicts the basis of protein-protein interaction (PPI) between peptide drug and GIRK4.
  • PPI protein-protein interaction
  • Molecular surface representation depicting different models of association (models 1-5) of peptide drug (red shades) with 350-374 region of GIRK4 C-terminus (navy blue). The 375- 399 region of GIRK 4 C-terminus is shown in gold.
  • Predicted structure of GIRK4 and peptidel were generated using l-TASSER.
  • Different models of PPI were generated using Threpp (model 1), COTH (models 3 and 4) and GRAMM (2 and 5).
  • Figure 19 depicts the effects of peptide-based drug on iPSC-aCMs. Immunoblotting following surface biotinylation of iPSC-aCMs treated with PBS (control) and peptide 1 showing reduced normalized GIRK4 membrane expression after peptide treatment.
  • homolog or “homologous” when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structure or to a high degree of similarity between the active site and the mechanism of action.
  • a homolog has a greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100 percent sequence identity).
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • variant and mutant when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.
  • conservative amino acid substitutions refers to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • conservative substitution is the exchange of one amino acid from a given group with a different amino acid from the same group.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).
  • term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical peptide sequences occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position.
  • the polypeptides GGGGGG and GGGGT have a sequence identity of 4 out of 5 or 80%.
  • the polypeptides GGGPPP and GGGAPPP have a sequence identity of 6 out of 7 or 85%.
  • any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent "similarity" is used to quantify the similarity between two sequences of the alignment.
  • substantially identical denotes a characteristic of a peptide sequence, wherein the peptide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 amino acid positions, frequently over a window of at least 15-30 amino acids, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the peptide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length sequences of the compositions claimed in the present disclosure.
  • the sequence of the active peptide will include at least 20 consecutive residues from Seq. ID NO: 1. In some implementations, the sequence will include at least 30 consecutive residues, at least 40 consecutive residues, at least 50 consecutive residues, or at least 60 consecutive residues, of Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 20-60 consecutive residues from Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 30-60 consecutive residues, from 30-40 consecutive residues, from 30-50 consecutive residues, from 40-50 consecutive residues, from 40-60 consecutive residues, or from 50-60 consecutive residues, from Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 20-40 or from 25-35 consecutive residues from Seq. ID NO: 1.
  • the active peptide will include a portion of Seq. ID NO: 1 taken from the C-terminus or near the C-terminus.
  • the active peptide will include a sequence having substantial identity or similarity to a portion of Seq. ID NO: 1 within the first 100 residues from the C-terminus, within the first 80 residues from the C- terminus, within the first 60 residues from the C-terminus, within the first 50 residues from the C-terminus, or within the first 40 residues from the C-terminus.
  • the active peptide can include a sequence corresponding to Seq. ID NO: 93. In some implementations, the active peptide can include a sequence having 90% or greater identity to Seq. ID NO: 93. In some implementations, the active peptide can include a sequence have 80% or greater identity, 85% or greater identity, 90% or greater identity, or 91% or greater identity to Seq. ID NO: 93.
  • the active peptide can include a sequence corresponding to Seq. ID NO: 94 or 95, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, ,90% or greater similarity, 92% or greater similarity to Seq. ID NO: 94. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 90% or greater similarity, 95% or greater similarity, or 97% or greater similarity to Seq. ID NO: 94 or 95.
  • X 1 is K and X 2 is N, Q, K, D, E, I, Y, or W. In some implementations, X 1 is E, R, Q, S, Y, or W, and X 2 is S.
  • the active peptide can include a sequence corresponding to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence having 90% or greater identity to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence have 80% or greater identity, 85% or greater identity, 95% or greater identity, or 98% or greater identity to Seq. ID NO: 94 or 95.
  • the therapeutic peptide can include a cell penetrating peptide ("CPP").
  • CPP cell penetrating peptide
  • the cell penetrating peptide will be conjugated to the active peptide.
  • the cell penetrating peptide can be a cationic cell penetrating peptide (for example a protamine, crotamine, maurocalcine, spider toxin Lycosin-I, BuCATHL4C, CB5005, or TD-34) a hydrophobic cell penetrating peptide, an amphiphatic cell penetrating peptide (from the PepFect family), or a combination of different cell penetrating peptides.
  • the therapeutic peptide consists of the active peptide, in that no additional peptide sequences are present.
  • the therapeutic peptide can include a cell penetrating peptide having at least 75% or greater similarity, 80% or greater similarity, 83% or greater similarity, 85% or greater similarity, 87% or greater similarity, 88% or greater similarity, at least 90% or greater similarity, 91% or greater similarity, 92% or greater similarity, 93% or greater similarity, 94% or greater similarity, 95% or greater similarity, 96% or greater similarity, or 97% or greater similarity, or 75% or greater identity, 80% or greater identity, 83% or greater identity, 85% or greater identity, 87% or greater identity, 88% or greater identity, at least 90% or greater identity, 91% or greater identity, 92% or greater identity, 93% or greater identity, 94% or greater identity, 95% or greater identity, 96% or greater identity, or 97% or greater identity to one of the following sequences:
  • ID NO 22 SQPEATKCFQWQRNM RKVRGPPVSCIKRDSPIQI Seq. ID NO 23 - GRGDSY Seq. ID NO 24 - (VRLPPP)3 Seq. ID NO 25 - KCFQWQRNMRKVRGPPVSCIKR Seq. ID NO 26 - RRIRPRPPRLPRPRPRPLPFPRPG Seq. ID NO 27 - RAGLQFPVG[RLLR]3 Seq. ID NO 28 - GLRKRLRKFRNKIKEK Seq. ID NO 29 - RGGRLCYCRRRFCVCVGR Seq. ID NO 30 - MVKSKIGSWILVLFVAMWSDVGLCKKRP Seq. ID NO 31 - DPRSFL Seq.
  • ID NO 32 NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ Seq.
  • ID NO 33 DAATATRGRSAASRPTERPRAPARSASRPRRPVE Seq.
  • ID NO 34 DPKGDPKGVTVTVTVTVTGKGDPKPD Seq.
  • ID NO 35 - PIEVCMYREP Seq.
  • ID NO 36 - CSIPPEVKFNKPFVYLI Seq.
  • ID NO 37 - LSTAADMQGVVTDGMASGLDKDYLKPDD Seq.
  • ID NO 38 - PFVYLI Seq.
  • ID NO 39 RLSGMNEVLSFRWL Seq.
  • ID NO 40 SDLWEMM MVSLACQY Seq. ID NO 41 - CRWRWKCCKK Seq.
  • ID NO 42 WLRRIKAWLRRIKALNRQLGVAA Seq.
  • ID NO 43 GLFGAIAGFIENGWEGMIDGWYG Seq.
  • ID NO 44 WEAKLAKALAKALAHLAKALAKALKACEA Seq.
  • ID NO 45 LSTAADMQGVVTDGMASGLDKDYLKPDD
  • the cell penetrating peptide can have substantial similarity or identity to GRKKRRQRRRPPQ (Seq. ID NO: 3) or PIEVCMYREP (Seq. ID NO: 35). In some implementations the cell penetrating peptide can have at least 90% identity, at least 95% identity, or at least 98% identity to one of Seq. ID NO: 3-92.
  • the cell penetrating peptide can have at least 90% identity, at least 84% identity, or at least 92% identity to Seq. ID NO: 3. In some implementations the cell penetrating peptide can have at least 80% identity, or at least 90% identity to Seq. ID NO: 35.
  • the cell penetrating peptide may be conjugated to the active portion via covalent bonds.
  • the cell penetrating peptide (e.g., Seq. ID NO: 3-92) may be conjugated to the active portion via a linker.
  • the linker can be a peptide linker or a non-peptidyl linker.
  • a peptidyl linker will consist solely of amino acid residues joined together by amide bonds, in which the linker is connected to both the active peptide and CPP via amide bonds.
  • a non-peptidyl linker will include non-amino acid moieties and nonamide linkages, although a non-peptidyl linker may include one or more amino acids and amide bonds as well. Such constructs are still considered therapeutic peptides for the purpose of this disclosure.
  • the linker is a peptide linker having the sequence -(G) X S- wherein x is from 3 to 10 (Seq. ID NO: 96). In some implementations, x is 4 (GGGGS Seq. ID NO: 97). In some implementations, the peptide linker has the formula (K) x wherein x is from 2-10 (Seq. ID NO: 98) or (Orn) x , wherein x is from 2-10 (Seq. ID NO: 99). In some implementations the peptide linker can be covalently joined to the C terminus of the active peptide and to the N terminus of the CPP.
  • non peptidyl linkers can be joined at or near the C terminus of the active peptide, and at or near N terminus of the CPP.
  • Non peptidyl linkers include those with bonds formed via click chemistry (e.g., alkyne/azide cycloaddition).
  • the C- terminus of the active peptide can be condensed with a carboxylic acid-reactive compound having an azide, for example methylamino-PEG x azide, amino-PEG x azide, aminooxy-PEG x azide, wherein x is 1-4.
  • the N terminus of the CPP may be condensed with an amine reactive alkyne containing compound (e.g., carboxylic acid, carboxylic acid derivative).
  • an amine reactive alkyne containing compound e.g., carboxylic acid, carboxylic acid derivative.
  • Cyclooctyne compounds such as DIBO, DBCO, and halogenated derivatives may also be used.
  • DIBO dihydroxybenzoctyne compounds
  • DBCO dibenzyl-N
  • the active peptide can include a sequence corresponding to any of Seq. ID NO: 100-107, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 95% or greater similarity, 96% or greater similarity, 97% or greater similarity, or 98% or greater similarity to any of Seq. ID NO: 100-107.
  • X 1 is K, E, R, Q, S, Y, or W
  • X 2 is S, N, Q, R, K, D, E, I, Y, or W.
  • X 1 is K and X 2 is N, Q, K, D, E, I, Y, or W.
  • X 1 is E, R, Q, S, Y, or W
  • X 2 is S.
  • the active peptide can include a sequence corresponding to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence having 90% or greater identity to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence having 80% or greater identity, 85% or greater identity, 95% or greater identity, 96% or greater identity, 97% or greater identity or 98% or greater identity to any of Seq. ID NO: 100-107.
  • compositions are formulated for parenteral administration and include one or more therapeutic peptides, dissolved or suspended in an acceptable carrier, such as an aqueous carrier.
  • an acceptable carrier such as an aqueous carrier.
  • aqueous carriers e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions can be sterilized by conventional sterilization techniques, including sterile filtered.
  • the resulting solutions may be packaged for use as is or they may lyophilized, for reconstitution by a medical provider.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • buffering agents such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the disclosure provides a composition that includes a therapeutic peptide of any of Seq. ID NO:1, 2, 93-95, or 100-107, an aqueous vehicle, and one or more pharmaceutically acceptable excipients.
  • the composition can be an aqueous or oil suspension prepared using a suitable dispersant or wetting agent and a suspending agent.
  • Injectable forms can be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent.
  • Acceptable solvents or vehicles include sterilized water, Ringer's solution, and isotonic aqueous saline solution.
  • Sterile oils can be employed as solvents, co-solvents, or suspending agents.
  • the oil is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.
  • the pharmaceutical formulation can be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates.
  • the formulations can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • the compounds can be formulated for parenteral administration by injection such as by bolus injection or continuous infusion.
  • a unit dosage form for injection can be in ampoules or in multi-dose containers.
  • the aqeuous composition can include a pharmaceutically acceptable buffer.
  • buffers include, without limitation, succinate buffers, acetate buffers, citrate buffers and phosphate buffers.
  • the aqeuous composition includes 0.1 mg/mL to 20 mg/mL of the therapeutic peptide and sodium chloride in an amount to give a substantially isotonic solution.
  • the therapeutic peptide can be present in a concentration of 1 mg/mL to 15 mg/mL or 2.5 mg/mL to 10 mg/mL.
  • the composition can be provided with instructions to reconstitute the composition using a volume of solvent sufficient to achieve the above concentrations.
  • therapeutic methods including the step of administering a therapeutic peptide to a subject in need thereof.
  • the method is for regulating IK.ACH activity and reducing AF inducibility by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein.
  • the method is for downregulating GIRK4 membrane expression by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein.
  • the method is for reducing the formation of GIRK tetramers by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein.
  • the method is for reducing the formation of abnormal GIRK4 homo-tetramers by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein.
  • a therapeutic peptide for example in a pharmaceutical formulation as described herein.
  • methods of treating a disorder including the step of administering a therapeutic peptide to a subject in need thereof.
  • the disorder is characterized by aberrant l K ,Ach activity and/or arrhythmic activity.
  • the disorder is characterized by upregulation of membrane GIRK4.
  • the disorder is characterized by excess formation of GIRK4 homo-tetramers.
  • Also disclosed herein are methods of treating a subject having a disorder characterized by an arrhythmia or structural heart disease leading to arrhythmia including the step of administering a therapeutic peptide to a subject in need thereof.
  • the subject is diagnosed with tachycardia, bradycardia, or a combination thereof.
  • the subject is diagnosed atrial fibrillation, atrial flutter, supraventricular tachycardia, sick sinus syndrome, atrioventricular dysfunction, tachy-brady syndrome, or a combination thereof.
  • the therapeutic peptides disclosed herein may be administered with one or more additional therapeutic agents.
  • the subject may also be administered a beta blocker, calcium channel blocker, sodium channel blocker, potassium channel blocker, or combination thereof.
  • the subject is administered atenolol, bisoprolol, carvedilol, metoprolol, propranolol, timolol, diltiazem, verapamil, digoxin, flecainide, propafenone, quinidine, amiodarone, sotalol, dofetilide, dronedarone, or a combination thereof.
  • l.TAT-Gl-C-terminus (peptide 1) GRKKRRQRRRPPQ-GGGGS- GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR (408-439) (Seq. ID NO: 102).
  • CPC-C-Gl-C-terminus (peptide 2) PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR (408-439) (Seq. ID NO: 103). Both peptides were synthesized by LifeTein. The peptide is composed of a CPP tag (TAT and CPC-C, respectively) and 408-439 region of GIRK1 C-terminus.
  • our custom peptide also contains a GGGGS linker to preserve the secondary structure of the 408-439 region while maintaining overall functionality.
  • the peptides underwent TFA removal (trifluoroacetic acid) for less toxicity to cells sensitive to TFA.
  • Our peptides have the same GIRK1 C-terminus sequence, and they have different delivery mechanisms to determine the most efficient delivery mechanism to the atrial myocytes.
  • TAT is a cationic CPP that induces direct penetration (low temperature and high concentration is recommended).
  • CPC-C is a hydrophobic CPP and is internalized through endocytosis (high temperature and low concentration). Both peptides were used at a concentration of 30pM and for 2-3 hours.
  • COTH CO-THreader
  • Threpp Multimeric Threading-based Protein-protein Interaction Predictor
  • GRAMM Global Range Molecular Matching
  • the peptide contacts the initial part of the 350-375 (blue) from inside, thereby invading the interface where the neighboring GIRK subunit would come to form a dimer.
  • the peptide contacts both 350-375 and 375-399 (Fig.18). Therefore, we hypothesize that the peptide would disrupt the self-assembly of GIRK4 homo-tetramer and its membrane localization.
  • hiPSC-CMs offer great potential for mechanism specific drug discovery and cardiotoxicity screenings in a human physiologically relevant model. They allow drug optimization, dosage adjustment, comparison between various drug targets and human validation before moving to the clinical setting.
  • iPSCs were differentiated into iPSC-aCMs as described by Cyganek et al., JCI Insight. 2018;3(12). Preliminary data for characterization, GIRK subunit expression and AP recordings are available in (Figs. 15- 16).
  • Preliminary immunoblotting studies using iPSCs differentiated to iPSC-aCMs showed reduced normalized GIRK4 membrane expression after treatment with peptide 1 following surface biotinylation (Fig.19).
  • the purpose of this model is only to identify the spatial locations of functionally important residues of C-terminus using both global and local structural frameworks of proteins.
  • the model with highest confidence score (C-score) was chosen for each and the accuracy of the model was further validated by structure-based superimposition with known GIRK2 structure (PDB-6XIS).
  • C-score structure-based superimposition with known GIRK2 structure
  • the C-terminus of both GIRKs is composed of stable alpha helix and structural variable loop regions.
  • GIRK4 C- terminus has an L shaped orientation with residues 375-399 arranged either in alpha helix or coil with 90° bent whereas the C-terminus of GIRK1 is arranged in U-shaped with one side facing the GIRK4 C-terminus.
  • GIRK1/4 hetero-tetramer and GIRK4 homo-tetramer are functional but not GIRK1 homo-tetramer
  • GIRK4 possesses the necessary information for cell surface localization of functional /K,ACH i.e. region 350-375 is responsible for its own surface localization either in hetero-tetramer with GIRK1 or as a homotetramer. Its distal region (375-399) presents a critical point of contact for GIRK1 surface localization in hetero-tetrameric channels.
  • GIRK subunits bind with FLOT-1 and GIRK and FLOT-1 are expressed in HL-1 cells.
  • Flotil lin-1 (FL0T1) is localized at the membrane and intercalated disc of cardiomyocytes. FL0T1 levels have been correlated with myocyte excitability and Na v 1.5 function. Previous research has suggested a role of FLOT-1 in Na v 1.5 trafficking.
  • FLOT-1 in Na v 1.5 trafficking.
  • Fig.5A-B control mouse atrial lysates
  • Fig.5C-E HL-1 cells are derived from the AT-1 mouse atrial cardiomyocyte tumor lineage and maintain the differentiated biochemical, morphological and electrophysiological properties. Therefore, HL-1 cells can be used as a model to evaluate changes in / K ,ACK-
  • GIRK1 and 4 subunits MUST form hetero-tetramers for proper/native /K.ACK regulation.
  • GIRK1 cannot form functional channels on its own, and instead requires association with GIRK4.
  • No detectable GIRK1 expression was observed at the membrane from atrial myocytes isolated from GIRK4 /_ mice.
  • GIRK4p.G387R +/+ mice are prone to AF upon atrial pacing.
  • Intracardiac programmed electrical stimulation (PES) studies were performed as described by Greer-Short et aL, Heart Rhythm. 2020;17(3):503-ll. Atrial and ventricular intracardiac electrograms were recorded using a catheter inserted via the right jugular vein. Surface and intracardiac ECG parameters were collected before and after AF inducibility. AF was induced using an atrial pacing protocol described in Greer-Short. Our data showed susceptibility of GIRK4p.G387R +/+ mice to AF upon atrial pacing compared to the control mice (Fig.11).
  • GIRK4p.G387R +/+ mice displayed heart rate variability at baseline. Genetic inactivation of GIRK4 in mice showed reduced heart rate variability characterized by reduced high frequency (HF) and low frequency (LF) parameters of heart rate variability spectra (HRV). In contrast, GIRK4p.G387R +/+ mice displayed increased HF and LF variability compared to control littermates with no changes in basal heart rate and RR interval ( Fig.12).
  • GIRK4p.G387R +/+ atrial myocytes displayed aberrant / K A ch fast desensitization.
  • hiPSC-aCMs Structural characterization of hiPSC-aCMs.
  • hiPSCs control line was generated by the Stanford iPSC Biobank.
  • hiPSCs were differentiated into ventricular cardiomyocytes (hiPSC-vCMs) via small moleculebased modulation of Wnt signaling, as described by Burridge et al., Nat Methods. 2014;ll(8):855-60 and Lian et aL, Nat Protoc. 2013;8(l):162-75.
  • Atrial subtype differentiation was performed as described by Cyganek.
  • hiPSC-vCMs generated under the control condition showed robust expression of ventricle-specific myosin light chain 2 (MLC2V) and lower levels of atrial MLC2A.
  • hiPSC-aCMs obtained from the RA-treated cultures revealed high expression of MLC2A, but no expression of MLC2V.
  • hiPSC-aCMs displayed increased expression of connexin-40 while hiPSC-vCMs displayed an increased expression of connexin-43 (Fig.15).
  • Action potential recordings from iPSC-aCMs displayed increased response of atrial myocyte to the modulator (carbachol, CCh).
  • CCh shortened APD.
  • iPSC-aCMs showed increased GIRK1 and GIRK4 protein levels compared to iPSC-vCMs.
  • KCNJ3 was identified to be highly expressed in (iPSC-aCMs) (Fig.16).
  • TAT-GIRKl-C-terminus peptide shows reduced /K.ACH compared to ML297 (/K AC activator) and control cells.
  • Mice lacking / K ,ACH via genetic inactivation were demonstrated to be resistant to CCh-induced AF.
  • CCh administration induced AF in 10 of 14 WT mice.
  • Such findings suggest that inhibition or abolishment of /K.ACH could be preventative against AF, thereby implicating a therapeutic role of K A ch inhibitors in the treatment of AF.
  • / K ,ACH was recorded in iPSC-aCMs.
  • / K ,ACH was characterized as a CCh induced current (Fig.17A).
  • a composition comprising an amino acid sequence, wherein said amino acid sequence comprises about 20-60 residues of SEQ. ID NO: 1, wherein the sequence is conjugated to a cell penetration peptide.
  • Aspect 2 The composition of aspect 1, wherein said amino acid sequence comprises residues at or near the C terminus of SEQ. ID NO: 1.
  • Aspect 3 The composition of aspect 1 or aspect 2, wherein the amino acid sequence comprises 90% or more identity to SEQ. ID NO: 2.
  • Aspect 4 The composition according to any one of aspects 1-3, wherein the cell penetration peptide comprises a cationic cell penetrating peptide, a hydrophobic cell penetrating peptide, or an amphiphatic cell penetrating peptide.
  • Aspect 5 The composition according to any one of aspects 1-4, wherein the cell penetrating peptide comprises any one of SEQ. ID NO: 3-92.
  • Aspect 6 The composition according to any one of aspects 1-5, wherein the cell penetrating peptide is selected from one of the following sequences:
  • ID NO 69 GALFLGWLGAAGSTM- GAPKKKRKV Seq.
  • ID NO 70 CHHHHHRRRRRR- RHHHHHC Seq.
  • ID NO 71 - ac-KWFETWFTEWPKKR- K-Cya Seq.
  • ID NO 72 - KKKK Seq.
  • ID NO 73 - ALFLGFLGAAGSTMGA- WSQPKKKRKV Seq.
  • ID NO 74 - RRRRRRRR Seq.
  • ID NO 75 - KKKKKKKK Seq.
  • ID NO 76 - KKKKKK Seq.
  • ID NO 77 - KKKKKKKKKKKK Seq.
  • ID NO 78 - KKKKKKKKKKKK Seq.
  • ID NO 88 KWLLRWLSRLLRWLAR WLG Seq.
  • ID NO 89 - LLWRLWRLLWRLWRLL Seq.
  • ID NO 90 KLALKLALKALKAALK- LA Seq.
  • ID NO 91 ALWKTLLKKVLKAPKK-KRKVC Seq.
  • ID NO 92 GWTLNSAGYLLGKINL- KALAALAKKIL
  • Aspect 7 The composition according to any one of aspects 1-6, wherein the amino acid sequence is conjugated to the cell penetrating peptide through a linker.
  • Aspect 8 The composition according to any one of aspects 1-7 wherein the amino acid sequence is conjugated to the cell penetrating peptide through a linker having the sequence - (G)xS-x, wherein x is from 3 to 10.
  • a composition comprising an amino acid sequence, wherein said amino acid sequence comprises 20-60 residues of SEQ. ID NO: 1.
  • Aspect 10 The composition of aspect 9, wherein said amino acid sequence comprises residues at or near the C terminus of SEQ. ID NO: 1.
  • Aspect 11 The composition of aspect 9 or aspect 10, wherein the amino acid sequence comprises 90% or more, 95% of more, or 98% or more identity to SEQ. ID NO: 2.
  • a composition comprising an amino acid sequence having the formula: GRKKRRQRRRPPQ-GGGGS- GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR.
  • a composition comprising an amino acid sequence having the formula: PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR
  • Aspect 14 The composition of any of aspects 1-13, wherein the composition is a lyophilized powder and optionally one or more pharmaceutically acceptable excipients.
  • Aspect 15 The composition of any of aspects 1-13, wherein the composition comprises water and optionally one or more pharmaceutically acceptable excipients.
  • Aspect 16 A method of regulating l K ,Ach activity and reducing AF inducibility, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 17 A method of downregulating GIRK4 membrane expression, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 18 A method of reducing the formation of GIRK tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 19 A method of reducing the formation of abnormal GIRK4 homo-tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 10 A method of treating a disorder characterized by aberrant l K ,Ach activity and arrhythmic activity, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 21 A method of treating a disorder characterized by upregulation of membrane GIRK4, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 22 A method of treating a disorder characterized by excess formation of GIRK4 homo-tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
  • Aspect 23 The method of any one of aspects 16-22, wherein the subject has a disorder characterized by an arrhythmia or structural heart disease leading to arrhythmia.
  • Aspect 24 The method of aspect 23, wherein the arrhythmia is tachycardia, bradycardia, or a combination thereof.
  • Aspect 25 The method of any one of aspects 23 or 24, wherein the arrhythmia is atrial fibrillation, atrial flutter, supraventricular tachycardia, sick sinus syndrome, atrioventricular dysfunction, tachy-brady syndrome, or a combination thereof.
  • Aspect 26 The method of any one of aspects 23-25, further administering to the subject a beta blocker, calcium channel blocker, sodium channel blocker, potassium channel blocker, or combination thereof.
  • Aspect 27 The method of any one of aspects 23-26, further administering to the subject atenolol, bisoprolol, carvedilol, metoprolol, propranolol, timolol, diltiazem, verapamil, digoxin, flecainide, propafenone, quinidine, amiodarone, sotalol, dofetilide, dronedarone, or a combination thereof.
  • compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims.
  • Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims.

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Abstract

Disclosed herein are therapeutic peptides useful for regulating IK,Ach activity and reducing AF inducibility. The peptides may be used to treat disorders characterized by abnormal IK,Ach activity and reducing AF inducibility, including atrial fibrillation.

Description

THERAPEUTIC PEPTIDES TO MANAGE ATRIAL FIBRILLATION
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of US Provisional Application 63/580,099, filed September 1, 2023, the contents of which are hereby incorporated in its entirety.
SEQUENCE LISTING
[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patentcenter encoded as XML in UTF-8 text. The electronic document, created on August 31, 2024, is entitled "103361-576WOl_ST26.xml", and is 142,973 bytes in size.
BACKGROUND
[0003] Atrial fibrillation (AF) is the most common sustained clinical arrhythmia with a lifetime incidence of up to 37% and is a major contributor to morbidity and mortality. AF increases risk of heart failure, myocardial infarction, chronic kidney disease, stroke, and death. Notably, it has been over 10 years since the introduction of any new therapeutic agent for AF. AF drug development has been diminished by many factors including limited efficacy and the risk of side effects. AF causes both electrical and structural remodeling of the atria. Both processes contribute to the gradual progression of AF and further increase the duration of the AF episode and to reduce the efficacy of antiarrhythmic drugs. Thus, there is the crucial need for development of anti-arrhythmic therapies that are atrial specific. There is particular interest in the K+ channels such as the acetylcholine-activated potassium channel ( KAch), formed of G- protein-gated inwardly rectifying potassium subunits (GIRK1 and GIRK4) and predominantly expressed in the atria to avoid ventricular pro-arrhythmic side effects.
[0004] GIRK4 inactivation has been proposed to target AF. Aberrant /K,Ach regulation is well documented and a major target for AF therapy. In fact, there are data to support that blocking lK,Ach can abrogate AF. The bee venom peptide, tertiapin, terminated pharmacologically and vagally induced AF in canines by blocking lK,Ach- Additionally, tertiapin successfully prevented and/or terminated adenosine induced AF in ex vivo human hearts. However, the tertiapin derivative, tertiapin Q, was shown to block other K+ channels and other GIRK hetero-tetramers in addition to the GIRK1/GIRK4. Other lK,Ach blockers have also been evaluated in previous studies but the field still lacks a targeted strategy. [0005] There remains a need for improved compositions and methods for the treatment of AF.
There remains a need for improved compositions and methods for selectively modulating GIRK4 expression and function. There remains a need for improved compositions and methods for blocking lK,Ach.
BRIEF DESCRIPTION OF THE FIGURES
[0006] Figure 1 depicts how normal atrial myocytes, l-GIRK4(blue)/GIRKl(red) assemble in the ER/trans-Golgi and then is delivered to the membrane. 2- and 3- In AF, there is less GIRK1 and increased formation of GIRK4 homo-tetramers at the membrane.
[0007] Figure 2 depicts a schematic presentation of predicted mode of action of GIRK1 C- terminus (408-439) peptide. The peptide disrupts the overall availability of free GIRK4 for GIRK4 homo-tetramer assembly by occupying the C-terminus in GIRK4 subunit. GIRK4 (blue), GIRK1 (red) and GIRK4p.G387R in black circle.
[0008] Figure 3 depicts the structure of modeled GIRK1/4 hetero-tetramer (Top Left). The C- terminal regions of GIRK1 (373-501, blue) and GIRK4 (375-399, yellow) critical for surface localization of GIRK1/4 hetero-tetramer stack closer to each other (Top Right). The GIRK1 region (408-439, red) makes critical contact with 375-399 region in GIRK4 (Bottom Left). Interaction between GIRK4 and GIRK1 is mediated by a H-bond between the side chain of lysine 426 in GIRK1 C-terminus and tyrosine 379 in GIRK4 C-terminus (Bottom Right). GIRK1 and GIRK4 models were generated using l-Tasser online tool and structural figures were made using Chimera.
[0009] Figure 4 depicts a comparison of channel pore size among GIRK4 homo-tetramer, GIRK1 homo-tetramer and GIRK1/4 hetero-tetramer. GIRK1 homo-tetramer showed significant constriction in pore opening on its intracellular side vs. extracellular side, thereby making it difficult for ions to pass. Pore opening towards extracellular and intracellular side is depicted in black and yellow circles, respectively.
[0010] Figure 5 depicts that GIRK subunits bind with Flotil lin-1 (FLOT-1) and expression of GIRK1, GIRK4 and FLOT-1 in HL-1 cells. Co-immunoprecipitation experiments were performed from mouse atrial lysates using (A) beads conjugated to GIRKl-lg or control-lg and (B) beads conjugated to GIRK4-lg or control-lg. Bound protein was immunoblotted with FLOT-1. (C-E) Expression of GIRK4 (C), GIRK1 (D) and FLOT-1 (E) in HL-1 cells. [0011] Figure 6 depicts aberrant expression of GIRK4 in dogs with persistent AF post chronic atrial pacing. (A-B) GIRK4 normalized to GAPDH in control and dogs post pacing, n=3/group.
[0012] Figure 7 depicts a GIRK4p.G387R+/+ mouse model. (A-B) Conserved sequence of GIRK4 binding motif across species. (C) Representative chromatograms of the GIRK4p.G387R+/+ mice carrying the point mutation at amino acid 387 and control littermates. (D) Kcnj5 relative expression in GIRK4p.G387R+/+ mice.
[0013] Figure 8 depicts immunoblotting of GIRK and FLOT-1 in GIRK4p.G387R+/+ mice. Immunoblots and quantitative analysis of normalized (A-B) GIRK1 protein expression, (C-D) GIRK4 and (E-F) FLOT-1 using GIRK4p.G387R+/+ atrial lysates compared to control lysates, n=4/genotype.
[0014] Figure 9 depicts GIRK1 and GIRK4 localization at the atrial myocytes. (A-B) Expression of GIRK1 at the membrane of atrial myocytes in WT (A, 20X; B, 63X mag). (C-D) GIRK4 is uniformly localized at membrane of atrial myocytes (Scale bars equal 10pm). (E) GIRK1 is uniformly expressed at the membrane of atrial myocytes in WT cells. (F) Lost/discontinuous expression of GIRK1 at the membrane of GIRK4p.G387R+/+ atrial myocyte. Scale bars equal 20pm.
[0015] Figure 10 depicts ECG findings in GIRK4p.G387R+/+ mice. No significant changes in (A) heart rate and (B) PR interval. (C-D) Mutant mice display a trending prolongation in Q.T and QTc intervals that did not reach statistical significance, n=10-15/group.
[0016] Figure 11 depicts that GIRK4p.G387R+/+ mice are more prone to atrial arrhythmias. Electrocardiogram representatives from programmed electric stimulation acquired from GIRK4p.G387R+/+ mice and their control littermates.
[0017] Figure 12 depicts GIRK4p.G387R+/+ mice display heart rate variability. GIRK4p.G387R+/+ mice show increased (A) High frequency (HF) and (B) Low frequency (LF) of heart rate variability (HRV) spectra. n=9-ll/group.
[0018] Figure 13 depicts that GIRK4p.G387R+/+ mice demonstrate a structural atrial phenotype. GIRK4-G387R+/+ mice displayed a (A) an increase in left atrial diameter, (B) no changes in left ventricular internal diameter end diastole (LVID, d), and (C) no changes in ejection fraction except a mild reduction at 20 wks., n=6/genotype.
[0019] Figure 14 depicts that GIRK4p.G387R+/+ atrial myocytes display aberrant lK,ACh fast desensitization. Representative IK.AC current traces from WT and GIRK4p.G387R+/+ atrial myocytes. Desensitization is expressed as ratio of IK.ACH (at t = 10s) over lK,ACh (peak) at the beginning. n=3 (mice) and 11 (cells). ****p<0.0001. [0020] Figure 15 depicts the characterization of hiPSC-CMs. Immunostaining of atrial and ventricular hiPSC-CMs for (A) MLC2a (green,), and MLC2v (red); and DAPI (blue for nuclei). Scale bars equal 125 pm. (B) sarcomeric a-actinin (green), and gap junction Cx-40, (red). (C) sarcomeric a-actinin (green), and gap junction Cx-43 (red). Scale bars (B-C) equal 75 pm.
[0021] Figure 16 depicts the electrical characterization of hiPSC-aCMs. (A) patch clamp recordings of action potentials (APs) from hiPSC-AMs ±10 pM CCh). CCh shortened APD. (B) Increased expression of GIRK1 and GIRK4 in hiPSC-aCMs vs. hiPSC-vCMs.
[0022] Figure 17 depicts the effects of peptide-based drug (A) Representative recordings from a control hiPSC-aCM. IK.AC (black trace) was quantified by subtraction of the current recorded without CCh (red trace) from the current recorded with CCh (blue trace). The insert shows the voltage protocol (B-C) Current-Voltage relationship for lK,ACh in control, ML297 (GIRK activator), and peptide (TAT-Gl-C-terminus) groups in hiPSC-aCMs. Two-way ANOVA analysis shows significant differences as indicated +p<0.001, #p<0.01 and *p<0.05. n=10 cells/group. (D-E) Immunoblotting following surface biotinylation of HL-1 cells treated with PBS (control), peptide 1 (TAT-Gl-C-terminus) and peptide 2 (CPC-C-Gl-C-terminus).
[0023] Figure 18 depicts the basis of protein-protein interaction (PPI) between peptide drug and GIRK4. Molecular surface representation depicting different models of association (models 1-5) of peptide drug (red shades) with 350-374 region of GIRK4 C-terminus (navy blue). The 375- 399 region of GIRK 4 C-terminus is shown in gold. Predicted structure of GIRK4 and peptidel were generated using l-TASSER. Different models of PPI were generated using Threpp (model 1), COTH (models 3 and 4) and GRAMM (2 and 5).
[0024] Figure 19 depicts the effects of peptide-based drug on iPSC-aCMs. Immunoblotting following surface biotinylation of iPSC-aCMs treated with PBS (control) and peptide 1 showing reduced normalized GIRK4 membrane expression after peptide treatment.
DETAILED DESCRIPTION
[0025] Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0026] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes- from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
[0027] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
[0028] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal embodiment. "Such as" is not used in a restrictive sense, but for explanatory purposes.
[0029] The term "homolog" or "homologous" when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structure or to a high degree of similarity between the active site and the mechanism of action. In a preferred embodiment, a homolog has a greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
[0030] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
[0031] The terms "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. One type of conservative substitution is the exchange of one amino acid from a given group with a different amino acid from the same group. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).
[0032] In certain embodiments, term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical peptide sequences occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
[0033] In certain embodiments, sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. For example, the polypeptides GGGGGG and GGGGT have a sequence identity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP and GGGAPPP have a sequence identity of 6 out of 7 or 85%. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent "similarity" is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: aromatic - F Y W; hydrophobic-A V I L; charged positive: R K H; charged negative - D E; polar - 8 T N Q.
[0034] The terms "substantial identity" as used herein denotes a characteristic of a peptide sequence, wherein the peptide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 amino acid positions, frequently over a window of at least 15-30 amino acids, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the peptide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length sequences of the compositions claimed in the present disclosure.
[0035] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
[0036] Disclosed herein are therapeutic peptides. In some implementations, the therapeutic peptide will include no more than 150 amino acid residues. In some implementations, the therapeutic peptide will include no more than 125 amino acid residues. In some implementations, the therapeutic peptide will include no more than 100 amino acid residues. In some implementations, the therapeutic peptide will include no more than 90 amino acid residues. In some implementations, the therapeutic peptide will include no more than 80 amino acid residues. In some implementations, the therapeutic peptide will include no more than 75 amino acid residues. [0037] The therapeutic peptide will include an active peptide, wherein the active peptide corresponds to at least a portion of amino acid sequence Seq. ID NO: 1.
Seq. ID NO: 1
MSALRRKFGDDYQVVTTSSSGSGLQPQGPGQDPQQQLVPKKKRQRFVDKNGRCNVQHGNLGSE TSRYLSDLFTTLVDLKWRWNLFIFILTYTVAWLFMASMWWVIAYTRGDLNKAHVGNYTPCVANVY NFPSAFLFFIETEATIGYGYRYITDKCPEGIILFLFQSILGSIVDAFLIGCMFIKMSQPKKRAETLMFSEHA VISM RDGKLTLM FRVGNLRNSHMVSAQIRCKLLKSRQTPEGEFLPLDQLELDVGFSTGADQLFLVSP LTICHVIDAKSPFYDLSQRSMQTEQFEIVVILEGIVETTGMTCQARTSYTEDEVLWGHRFFPVISLEEG FFKVDYSQFHATFEVPTPPYSVKEQEEMLLMSSPLIAPAITNSKERHNSVECLDGLDDITTKLPSKLQK ITGREDFPKKLLRMSSTTSEKAYSLGDLPMKLQRISSVPGNSEEKLVSKTTKM LSDPMSQSVADLPPK LQKMAGGAARM EGNLPAKLRKMNSDRFT
[0038] In some implementations, the sequence of the active peptide will include at least 20 consecutive residues from Seq. ID NO: 1. In some implementations, the sequence will include at least 30 consecutive residues, at least 40 consecutive residues, at least 50 consecutive residues, or at least 60 consecutive residues, of Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 20-60 consecutive residues from Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 30-60 consecutive residues, from 30-40 consecutive residues, from 30-50 consecutive residues, from 40-50 consecutive residues, from 40-60 consecutive residues, or from 50-60 consecutive residues, from Seq. ID NO: 1. In some implementations, the sequence of the active peptide will include from 20-40 or from 25-35 consecutive residues from Seq. ID NO: 1.
[0039] In certain implementations, the active peptide will include a portion of Seq. ID NO: 1 taken from the C-terminus or near the C-terminus. In some implementations, the active peptide will include a sequence having substantial identity or similarity to a portion of Seq. ID NO: 1 within the first 100 residues from the C-terminus, within the first 80 residues from the C- terminus, within the first 60 residues from the C-terminus, within the first 50 residues from the C-terminus, or within the first 40 residues from the C-terminus.
[0040] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 2, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to Seq. ID NO: 2. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 95% or greater similarity, or 96% or greater similarity to Seq. ID NO: 2
Seq. ID NO: 2 - GREDFPKKLLRMSSTTSEKAYSLGDLPM KLQR [0041] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 2. In some implementations, the active peptide can include a sequence having 90% or greater identity to Seq. ID NO: 2. In some implementations, the active peptide can include a sequence have 80% or greater identity, 85% or greater identity, 95% or greater identity, or 96% or greater identity to Seq. ID NO: 2.
[0042] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 93, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to Seq. ID NO: 93. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 90% or greater similarity, or 91% or greater similarity to Seq. ID NO: 93 Seq. ID NO: 93 - RMSSTTSEKAYS
[0043] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 93. In some implementations, the active peptide can include a sequence having 90% or greater identity to Seq. ID NO: 93. In some implementations, the active peptide can include a sequence have 80% or greater identity, 85% or greater identity, 90% or greater identity, or 91% or greater identity to Seq. ID NO: 93.
[0044] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 94 or 95, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, ,90% or greater similarity, 92% or greater similarity to Seq. ID NO: 94. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 90% or greater similarity, 95% or greater similarity, or 97% or greater similarity to Seq. ID NO: 94 or 95.
Seq. ID NO: 94 - GREDFPKKLLRMSSTTSEX1AYX2LGDLPMKLQR
Seq. ID NO: 95 - RMSSTTSEX^YX2 where X1 is K, E, R, Q, S, Y, or W, and X2 is S, N, Q, R, K, D, E, I, Y, or W.
[0045] In some implementations, X1 is K and X2 is N, Q, K, D, E, I, Y, or W. In some implementations, X1 is E, R, Q, S, Y, or W, and X2 is S.
[0046] In some implementations, the active peptide can include a sequence corresponding to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence having 90% or greater identity to Seq. ID NO: 94 or 95. In some implementations, the active peptide can include a sequence have 80% or greater identity, 85% or greater identity, 95% or greater identity, or 98% or greater identity to Seq. ID NO: 94 or 95.
[0047] In some implementations, the therapeutic peptide can include a cell penetrating peptide ("CPP"). In some implementations, the cell penetrating peptide will be conjugated to the active peptide. In some implementations, the cell penetrating peptide can be a cationic cell penetrating peptide (for example a protamine, crotamine, maurocalcine, spider toxin Lycosin-I, BuCATHL4C, CB5005, or TD-34) a hydrophobic cell penetrating peptide, an amphiphatic cell penetrating peptide (from the PepFect family), or a combination of different cell penetrating peptides.
[0048] In some implementations, the therapeutic peptide consists of the active peptide, in that no additional peptide sequences are present. In some implementations, the therapeutic peptide can include a cell penetrating peptide having at least 75% or greater similarity, 80% or greater similarity, 83% or greater similarity, 85% or greater similarity, 87% or greater similarity, 88% or greater similarity, at least 90% or greater similarity, 91% or greater similarity, 92% or greater similarity, 93% or greater similarity, 94% or greater similarity, 95% or greater similarity, 96% or greater similarity, or 97% or greater similarity, or 75% or greater identity, 80% or greater identity, 83% or greater identity, 85% or greater identity, 87% or greater identity, 88% or greater identity, at least 90% or greater identity, 91% or greater identity, 92% or greater identity, 93% or greater identity, 94% or greater identity, 95% or greater identity, 96% or greater identity, or 97% or greater identity to one of the following sequences:
Seq. ID NO 3 - GRKKRRQRRRPPQ Seq. ID NO 4 - YGRKKRRQRRR Seq. ID NO 5 - CYGRKKRRQRRR Seq. ID NO 6 - RKKRRQRRR Seq. ID NO 7 - TRQARRNRRRRWRERQR Seq. ID NO 8 - RQIKIWFQNRRMKWKK Seq. ID NO 9 - LLIILRRRIRKQAHAHSK Seq. ID NO 10 - MVTVLFRRLRIRRACGPPRVRV Seq. ID NO 11 - MVRRFLVTLRIRRACGPPRVRV Seq. ID NO 12 - INLKALAALAKKIL Seq. ID NO 13 - AGYLLGKINLKALAALAKKIL Seq. ID NO 14 - CGYGPKKKRKVGG Seq. ID NO 15 - PKKKRKV Seq. ID NO 16 - KWRRKLKKLRPKKKRKV Seq. ID NO 17 - KWRRKLKKLR Seq. ID NO 18 - LGTYTQDFNKFHTFPQTAIGVGAP Seq. ID NO 19 - RKKRRRESRKKRRRES Seq. ID NO 20 - HSDGTFTSELSRLRDSARLQRLLQGLV Seq. ID NO 21 - LLGDFFRKSKEKIGKE FKRIVQRIKDFLRN LVPRTES Seq. ID NO 22 - SQPEATKCFQWQRNM RKVRGPPVSCIKRDSPIQI Seq. ID NO 23 - GRGDSY Seq. ID NO 24 - (VRLPPP)3 Seq. ID NO 25 - KCFQWQRNMRKVRGPPVSCIKR Seq. ID NO 26 - RRIRPRPPRLPRPRPRPLPFPRPG Seq. ID NO 27 - RAGLQFPVG[RLLR]3 Seq. ID NO 28 - GLRKRLRKFRNKIKEK Seq. ID NO 29 - RGGRLCYCRRRFCVCVGR Seq. ID NO 30 - MVKSKIGSWILVLFVAMWSDVGLCKKRP Seq. ID NO 31 - DPRSFL Seq. ID NO 32 - NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ Seq. ID NO 33 - DAATATRGRSAASRPTERPRAPARSASRPRRPVE Seq. ID NO 34 - DPKGDPKGVTVTVTVTVTGKGDPKPD Seq. ID NO 35 - PIEVCMYREP Seq. ID NO 36 - CSIPPEVKFNKPFVYLI Seq. ID NO 37 - LSTAADMQGVVTDGMASGLDKDYLKPDD Seq. ID NO 38 - PFVYLI Seq. ID NO 39 - RLSGMNEVLSFRWL Seq. ID NO 40 - SDLWEMM MVSLACQY Seq. ID NO 41 - CRWRWKCCKK Seq. ID NO 42 - WLRRIKAWLRRIKALNRQLGVAA Seq. ID NO 43 - GLFGAIAGFIENGWEGMIDGWYG Seq. ID NO 44 - WEAKLAKALAKALAHLAKALAKALKACEA Seq. ID NO 45 - LSTAADMQGVVTDGMASGLDKDYLKPDD
Seq. ID NO 46 - PIEVCMYREP Seq. ID NO 47 - VPTLK Seq. ID NO 48 - RRQRRTSKLMKR Seq. ID NO 49 - KGRKKRRQRRRPPQ Seq. ID NO 50 - RRRRNRTRRNRRRVR-amide Seq. ID NO 51 - Acetyl-KLALKLALKALKAALKLA-amide Seq. ID NO 52 - PLSSIFSRIGDP Seq. ID NO 53 - RPKPQQFGLM-amide Seq. ID NO 54 - YKQSHKKGGKKGSG Seq. ID NO 55 - RRRRRRRRR Seq. ID NO 56 - GLFKALLKLLKSLWKLLLKA Seq. ID NO 57 - WEAKLAKALAKALAKHLAKALAKALKACEA Seq. ID NO 58 - RQIRIWFQNRRMRWRR Seq. ID NO 59 - RRRRRRHHHH Seq. ID NO 60 - GLWRALWRLLRSLWRLLWRA Seq. ID NO 61 - KAFAKLAARLYRKALARQLGVAA Seq. ID NO 62 - KETWWETWWTEWSQPKKKRKVCya Seq. ID NO 63 - GLFRALLRLLRSLWRLLLRA Seq. ID NO 64 - RAGLQFPVGRLLRRLLR Seq. ID NO 65 - RRRR Seq. ID NO 66 - RRRRRR Seq. ID NO 67 - RRRRRRRRRR Seq. ID NO 68 - RRRRRRRRRRRR Seq. ID NO: 69 - GALFLGWLGAAGSTMGAPKKKRKV
Seq. ID NO: 70 - CHHHHHRRRRRRRRRHHHHHC
Seq. ID NO: 71 - ac-KWFETWFTEWPKKRKCya
Seq. ID NO: 72 - KKKK
Seq. ID NO: 73 - ALFLGFLGAAGSTMGAWSQPKKKRKV
Seq. ID NO: 74 - RRRRRRRR
Seq. ID NO: 75 - KKKKKKKK
Seq. ID NO: 76 - KKKKKK
Seq. ID NO: 77 - KKKKKKKKKK
Seq. ID NO: 78 - KKKKKKKKKKKK
Seq. ID NO: 79 - RRQRR
Seq. ID NO: 80 - RRQRRQRR
Seq. ID NO: 81 - RRQRRQRRQRR
Seq. ID NO: 82 - GALFLGFLGAAGSTMGAWSQPKSKRKV
Seq. ID NO: 83 - RRRRRRRRRRRRRRR
Seq. ID NO: 84 - HHHHHHHHRRRRRRRRRRRRRRR
Seq. ID NO: 85 - HHHHHHHHHHHHHHHHRRRRRRRRRRRRRRR
Seq. ID NO: 86 - ISFDELLDYYGESGS
Seq. ID NO: 87 - GYGYGYGYGYGYGYGYKKRKKRKKRKKRKQQKQQKRRK
Seq. ID NO: 88 - KWLLRWLSRLLRWLARWLG
Seq. ID NO: 89 - LLWRLWRLLWRLWRLL
Seq. ID NO: 90 - KLALKLALKALKAALKLA
Seq. ID NO: 91 - ALWKTLLKKVLKAPKKKRKVC
Seq. ID NO: 92 - GWTLNSAGYLLGKINLKALAALAKKIL
Cya = cysteamine
[0049] In some implementations the cell penetrating peptide can have substantial similarity or identity to GRKKRRQRRRPPQ (Seq. ID NO: 3) or PIEVCMYREP (Seq. ID NO: 35). In some implementations the cell penetrating peptide can have at least 90% identity, at least 95% identity, or at least 98% identity to one of Seq. ID NO: 3-92.
[0050] In some implementations the cell penetrating peptide can have at least 90% identity, at least 84% identity, or at least 92% identity to Seq. ID NO: 3. In some implementations the cell penetrating peptide can have at least 80% identity, or at least 90% identity to Seq. ID NO: 35.
[0051] In some implementations, the cell penetrating peptide may be conjugated to the active portion via covalent bonds. The cell penetrating peptide (e.g., Seq. ID NO: 3-92) may be conjugated to the active portion via a linker. The linker can be a peptide linker or a non-peptidyl linker. As used herein, a peptidyl linker will consist solely of amino acid residues joined together by amide bonds, in which the linker is connected to both the active peptide and CPP via amide bonds. A non-peptidyl linker, on the other hand, will include non-amino acid moieties and nonamide linkages, although a non-peptidyl linker may include one or more amino acids and amide bonds as well. Such constructs are still considered therapeutic peptides for the purpose of this disclosure.
[0052] In some implementations, the linker is a peptide linker having the sequence -(G)XS- wherein x is from 3 to 10 (Seq. ID NO: 96). In some implementations, x is 4 (GGGGS Seq. ID NO: 97). In some implementations, the peptide linker has the formula (K)x wherein x is from 2-10 (Seq. ID NO: 98) or (Orn)x, wherein x is from 2-10 (Seq. ID NO: 99). In some implementations the peptide linker can be covalently joined to the C terminus of the active peptide and to the N terminus of the CPP. In certain implementations, non peptidyl linkers can be joined at or near the C terminus of the active peptide, and at or near N terminus of the CPP. Non peptidyl linkers include those with bonds formed via click chemistry (e.g., alkyne/azide cycloaddition). The C- terminus of the active peptide can be condensed with a carboxylic acid-reactive compound having an azide, for example methylamino-PEGxazide, amino-PEGxazide, aminooxy-PEGxazide, wherein x is 1-4. The N terminus of the CPP may be condensed with an amine reactive alkyne containing compound (e.g., carboxylic acid, carboxylic acid derivative). Cyclooctyne compounds, such as DIBO, DBCO, and halogenated derivatives may also be used. The skilled person understands the opposite connectivity may be achieved using differently functionalized azide and alkyne compounds.
[0053] In some implementations, the active peptide can include a sequence corresponding to any of Seq. ID NO: 100-107, or variant thereof. In some implementations, the active peptide can include a sequence having 90% or greater similarity to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence have 80% or greater similarity, 85% or greater similarity, 95% or greater similarity, 96% or greater similarity, 97% or greater similarity, or 98% or greater similarity to any of Seq. ID NO: 100-107.
Seq. ID NO: 100 - GRKKRRQRRRPPQ-GGGGS-RMSSTTSEKAYS
Seq. ID NO: 101 - PIEVCMYREP-GGGGS- RMSSTTSEKAYS
Seq. ID NO: 102 - GRKKRRQRRRPPQ-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR Seq. ID NO: 103 - PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR Seq. ID NO: 104 - GRKKRRQRRRPPQ-GGGGS- RMSSTTSEX2AYX2 Seq. ID NO: 105 - PIEVCMYREP-GGGGS- RMSSTTSEX^YX2
Seq. ID NO: 106 - GRKKRRQRRRPPQ-GGGGS-GREDFPKKLLRMSSTTSEX1AYX2LGDLPMKLQR Seq. ID NO: 107 - PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEX1AYX2LGDLPMKLQR where X1 is K, E, R, Q, S, Y, or W, and X2 is S, N, Q, R, K, D, E, I, Y, or W. [0054] In some implementations, X1 is K and X2 is N, Q, K, D, E, I, Y, or W. In some implementations, X1 is E, R, Q, S, Y, or W, and X2 is S.
[0055] In some implementations, the active peptide can include a sequence corresponding to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence having 90% or greater identity to any of Seq. ID NO: 100-107. In some implementations, the active peptide can include a sequence having 80% or greater identity, 85% or greater identity, 95% or greater identity, 96% or greater identity, 97% or greater identity or 98% or greater identity to any of Seq. ID NO: 100-107.
[0056] Also disclosed herein are pharmaceutical compositions including the therapeutic peptides disclosed herein. In some embodiments, the peptides and compositions may be administered by any suitable route of administration, including, but not limited to, injection (subcutaneous, intraperitoneal, intravenous, intrathecal, intramuscular, intracerebroventricular, and spinal injection), intranasal, oral, transdermal, parenteral, inhalation, nasopharyngeal or transmucosal absorption. Sustained release formulations and methods for targeted delivery are known in the art and include, for example, use of liposomes, drug loaded biodegradable microspheres, drug-polymer conjugates, drug-specific binding agent conjugates and the like.
[0057] In some implementations the compositions are formulated for parenteral administration and include one or more therapeutic peptides, dissolved or suspended in an acceptable carrier, such as an aqueous carrier. Any variety of aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional sterilization techniques, including sterile filtered. The resulting solutions may be packaged for use as is or they may lyophilized, for reconstitution by a medical provider. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0058] In some implementations, the disclosure provides a composition that includes a therapeutic peptide of any of Seq. ID NO:1, 2, 93-95, or 100-107, an aqueous vehicle, and one or more pharmaceutically acceptable excipients. In some implementations the composition can be an aqueous or oil suspension prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms can be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, and isotonic aqueous saline solution. Sterile oils can be employed as solvents, co-solvents, or suspending agents. Generally, the oil is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.
[0059] For injection, the pharmaceutical formulation can be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The compounds can be formulated for parenteral administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection can be in ampoules or in multi-dose containers.
[0060] In some implementations, the aqeuous composition can include a pharmaceutically acceptable buffer. Such buffers include, without limitation, succinate buffers, acetate buffers, citrate buffers and phosphate buffers.
[0061] In some implementations, the aqeuous composition includes 0.1 mg/mL to 20 mg/mL of the therapeutic peptide and sodium chloride in an amount to give a substantially isotonic solution. In some implementations, the therapeutic peptide can be present in a concentration of 1 mg/mL to 15 mg/mL or 2.5 mg/mL to 10 mg/mL. When the composition is provided as a lyophilized powder, the composition can be provided with instructions to reconstitute the composition using a volume of solvent sufficient to achieve the above concentrations.
[0062] Also disclosed herein are therapeutic methods including the step of administering a therapeutic peptide to a subject in need thereof. In some implementations the method is for regulating IK.ACH activity and reducing AF inducibility by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein. In some implementations the method is for downregulating GIRK4 membrane expression by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein. In some implementations the method is for reducing the formation of GIRK tetramers by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein. In some implementations the method is for reducing the formation of abnormal GIRK4 homo-tetramers by administering to a subject in need thereof a therapeutic peptide, for example in a pharmaceutical formulation as described herein. [0063] Also disclosed herein are methods of treating a disorder including the step of administering a therapeutic peptide to a subject in need thereof. In some implementations the disorder is characterized by aberrant lK,Ach activity and/or arrhythmic activity. In some implementations the disorder is characterized by upregulation of membrane GIRK4. In some implementations the disorder is characterized by excess formation of GIRK4 homo-tetramers.
[0064] Also disclosed herein are methods of treating a subject having a disorder characterized by an arrhythmia or structural heart disease leading to arrhythmia including the step of administering a therapeutic peptide to a subject in need thereof. In some implementations, the subject is diagnosed with tachycardia, bradycardia, or a combination thereof. In some implementations, the subject is diagnosed atrial fibrillation, atrial flutter, supraventricular tachycardia, sick sinus syndrome, atrioventricular dysfunction, tachy-brady syndrome, or a combination thereof.
[0065] The therapeutic peptides disclosed herein may be administered with one or more additional therapeutic agents. For example, in addition to the therapeutic peptide, the subject may also be administered a beta blocker, calcium channel blocker, sodium channel blocker, potassium channel blocker, or combination thereof. In some implementations, the subject is administered atenolol, bisoprolol, carvedilol, metoprolol, propranolol, timolol, diltiazem, verapamil, digoxin, flecainide, propafenone, quinidine, amiodarone, sotalol, dofetilide, dronedarone, or a combination thereof.
EXAMPLES
[0066] The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.
Peptide Synthesis and Mode of Action:
[0067] l.TAT-Gl-C-terminus (peptide 1) GRKKRRQRRRPPQ-GGGGS- GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR (408-439) (Seq. ID NO: 102). 2. CPC-C-Gl-C-terminus (peptide 2) PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR (408-439) (Seq. ID NO: 103). Both peptides were synthesized by LifeTein. The peptide is composed of a CPP tag (TAT and CPC-C, respectively) and 408-439 region of GIRK1 C-terminus. In addition, our custom peptide also contains a GGGGS linker to preserve the secondary structure of the 408-439 region while maintaining overall functionality. The peptides underwent TFA removal (trifluoroacetic acid) for less toxicity to cells sensitive to TFA. Our peptides have the same GIRK1 C-terminus sequence, and they have different delivery mechanisms to determine the most efficient delivery mechanism to the atrial myocytes. TAT is a cationic CPP that induces direct penetration (low temperature and high concentration is recommended). CPC-C is a hydrophobic CPP and is internalized through endocytosis (high temperature and low concentration). Both peptides were used at a concentration of 30pM and for 2-3 hours.
[0068] • Mode of action of the peptide drug: We modeled the expected configuration of the peptide using l-TASSER server. The modeled peptide has a U-shaped conformation, which retains the original secondary organization of the 419-428 region, similar to the identified GIRK1 C-terminus (Fig.18). However, the attached CPP tag (GRKKRRQRRRPPQ) and -GGGGS linker changes the original morphology of the identified GIRK1 region by further stabilizing its flexible loops i.e. 408-418, 429-439 in the identified GIRK1 region. To predict the 3D model of proteinprotein interaction (PPI) complex, between the peptide drug and GIRK4 protein, we used numerous algorithms. First, COTH (CO-THreader), a multiple-chain protein-threading algorithm, is designed to identify and recombine protein complex structures from both tertiary and complex structure libraries. Second, Threpp (Multimeric Threading-based Protein-protein Interaction Predictor) is a computational algorithm for PPI prediction, and finally, GRAMM (Global Range Molecular Matching)- a web server for protein docking which predicts a spectrum of docking poses that characterize the intermolecular energy landscape in protein interaction. In models 1, 3, and 4, the drug (red) lies closer to the distal GIRK4 (375-399, gold) and binds to the proximal GIRK4 C-terminus (350-375, blue). In model 5, the peptide contacts the initial part of the 350-375 (blue) from inside, thereby invading the interface where the neighboring GIRK subunit would come to form a dimer. Lastly, in model 2, the peptide contacts both 350-375 and 375-399 (Fig.18). Therefore, we hypothesize that the peptide would disrupt the self-assembly of GIRK4 homo-tetramer and its membrane localization.
[0069] hiPSC-CMs offer great potential for mechanism specific drug discovery and cardiotoxicity screenings in a human physiologically relevant model. They allow drug optimization, dosage adjustment, comparison between various drug targets and human validation before moving to the clinical setting. We validated the impact of peptide-based drug delivery on GIRK4 membrane expression in hiPSC-aCMs. We decided to compare the effects of hiPSC-aCMs treated with peptide to control hiPSC-aCMs (treated with PBS). iPSCs were differentiated into iPSC-aCMs as described by Cyganek et al., JCI Insight. 2018;3(12). Preliminary data for characterization, GIRK subunit expression and AP recordings are available in (Figs. 15- 16). Preliminary immunoblotting studies using iPSCs differentiated to iPSC-aCMs showed reduced normalized GIRK4 membrane expression after treatment with peptide 1 following surface biotinylation (Fig.19).
[0070] Identification of the region 408-439 in GIRK1 C-terminus for peptide design. Despite having high level of sequence similarity among GIRK proteins, only GIRK2 structure has been characterized. We modeled the predicted protein structure of GIRK1 and GIRK4 using a composite approach which involves combination of 1) comparative modeling (for evolutionary related homologous template), 2) threading (evolutionary unrelated proteins) and 3) ab initio modeling (modeling from scratch) using an online available tool- l-TASSER (iterative threading assembly refinement). The relationship between structure and function is complex, as many protein folds/families are known to be functionally promiscuous while different folds can perform the same function. Therefore, the purpose of this model is only to identify the spatial locations of functionally important residues of C-terminus using both global and local structural frameworks of proteins. The model with highest confidence score (C-score) was chosen for each and the accuracy of the model was further validated by structure-based superimposition with known GIRK2 structure (PDB-6XIS). In our predicted hetero-tetrameric model, the C-terminus of both GIRKs is composed of stable alpha helix and structural variable loop regions. GIRK4 C- terminus has an L shaped orientation with residues 375-399 arranged either in alpha helix or coil with 90° bent whereas the C-terminus of GIRK1 is arranged in U-shaped with one side facing the GIRK4 C-terminus. The lysine 426 in GIRK1 C-terminus makes H bond with the side chain of tyrosine 379 in GIRK4 C-terminus suggesting that secondary structure (419-428) in GIRK1 terminus makes critical contact with GIRK4 C-terminus (375-399). However, we focus on the region 408-439 due to inherit flexibility of the C-terminal region loop and for stabilizing 419-428 secondary structures (Fig.3).
[0071] • GIRK1/4 hetero-tetramer and GIRK4 homo-tetramer are functional but not GIRK1 homo-tetramer Previous research has demonstrated that GIRK1 cannot form a functional KAch channel by itself and instead requires GIRK4 for membrane localization. GIRK4 possesses the necessary information for cell surface localization of functional /K,ACH i.e. region 350-375 is responsible for its own surface localization either in hetero-tetramer with GIRK1 or as a homotetramer. Its distal region (375-399) presents a critical point of contact for GIRK1 surface localization in hetero-tetrameric channels. Our computational studies have also showed that the organization of long GIRK1 C-terminus in GIRK1 homo-tetrameric model would block the efficient ion movement by limiting the channel pore size towards the intracellular side. The channel pore size in both hetero-tetramer and homo-tetramer is undisturbed and can allow the passage of ions (Fig.4).
[0072] • GIRK subunits bind with FLOT-1 and GIRK and FLOT-1 are expressed in HL-1 cells.
Flotil lin-1 (FL0T1) is localized at the membrane and intercalated disc of cardiomyocytes. FL0T1 levels have been correlated with myocyte excitability and Nav1.5 function. Previous research has suggested a role of FLOT-1 in Nav1.5 trafficking. Here, we confirm the interaction of both GIRK1 and GIRK4 with FLOT-1 using control mouse atrial lysates (Fig.5A-B). We also confirm the expression of GIRK1, GIRK4 and FLOT-1 in HL-1 cells (Fig.5C-E). HL-1 cells are derived from the AT-1 mouse atrial cardiomyocyte tumor lineage and maintain the differentiated biochemical, morphological and electrophysiological properties. Therefore, HL-1 cells can be used as a model to evaluate changes in /K,ACK-
[0073] • Aberrant expression of GIRK subunits in AF. Previous work showed alteration/reduction in protein levels of Kv4.3, Kv1.5 and Kir3.1 (GIRK1) in patients with persistent AF. In addition, right atrial tissues from dogs following chronic atrial pacing to induce persistent AF showed increased GIRK4 protein levels (Fig.6).
[0074] • Validation and Kcnj5 relative expression in GIRK4p.G387R+/+ mouse, / .ACK is tightly regulated by the assembly and targeting of channel hetero-tetramers comprised of 2 GIRK1 and 2 GIRK4 subunits. /K,AC can exist either as a hetero-tetramer comprised of two GIRK1 and two GIRK4 or as a homo-tetramer of four GIRK4. However, the expression of only GIRK4 in atrial myocytes resulted in a loss of acute desensitization, a reduction of /K. CK, and a slowing of current activation. Therefore, GIRK1 and 4 subunits MUST form hetero-tetramers for proper/native /K.ACK regulation. GIRK1 cannot form functional channels on its own, and instead requires association with GIRK4. No detectable GIRK1 expression was observed at the membrane from atrial myocytes isolated from GIRK4 /_mice. These data support the role of GIRK4 for proper processing and localization of GIRK1 in a functional hetero-tetramer.
Dysfunction of /K.AC in humans and animal models result in arrhythmia including AF. The field has actually identified a critical motif (375-399) on GIRK4 C-terminus required for regulation of the KACK localization at the membrane. Our preliminary data show aberrant /K.ACK activity in the mice carrying the GIRK4p.G387R variant. This variant is interestingly located in the motif identified on GIRK4. Human GIRK4p.G387R (Fig.7) variant is reported as a genetic cause for inherited arrhythmia including AF and LQT. It is noteworthy that human phenotypes do not match GIRK4 /_ mouse phenotypes. Instead, human phenotypes are consistent with models that results in decreased GIRK1/4 heterotetramer targeting. However, GIRK4 homo-tetramers with 'dysregulated' / ,AC (a known property of GIRK4 improperly assembled homo-tetramers), result in significant heterogeneity in atrial action potential duration (APD), particularly in response to parasympathetic stimulation. We have created a knock in model harboring a point mutation in the GIRK4 C-terminus motif 375-399 at amino acid 387 (GIRK4p.G387R+/+). These mice were confirmed by sequencing analysis, and they showed an increase in Kcnj5 relative expression (Fig-7).
[0075] • Expression and localization of GIRK subunits in GIRK4p.G387R+/+ mouse. In our mouse model harboring the point variant (GIRK4p.G387R+/+), there was a significant upregulation of both GIRK1 and GIRK4 protein levels in the atrial lysates combined with a significant reduction in FLOT-1 (Fig.8). In normal atrial sections, both GIRK1 and GIRK4 are localized at the membrane of atrial myocytes. Immunostaining data using atrial myocytes isolated from GIRK4p.G387R+/+ and control mice showed the localization of GIRK1 at the membrane of control atrial cells consistent with the expression of the membrane marker wheat germ agglutinin (WGA). However, the GIRK4p.G387R+/+ atrial myocytes showed loss of GIRK1 localization at the membrane (Fig.9).
[0076] • No significant ECG changes in GIRK4p.G387R+/+ mouse. Patients harboring the p.G387R variant showed a mild prolonged corrected QT interval and AF was also reported by these patients. Our recordings from conscious GIRK4p.G387R+/+ mice did not show changes in heart rate or PR interval. Interestingly, QT and QTc were not significantly different across genotypes (Fig.10).
[0077] • GIRK4p.G387R+/+ mice are prone to AF upon atrial pacing. Intracardiac programmed electrical stimulation (PES) studies were performed as described by Greer-Short et aL, Heart Rhythm. 2020;17(3):503-ll. Atrial and ventricular intracardiac electrograms were recorded using a catheter inserted via the right jugular vein. Surface and intracardiac ECG parameters were collected before and after AF inducibility. AF was induced using an atrial pacing protocol described in Greer-Short. Our data showed susceptibility of GIRK4p.G387R+/+ mice to AF upon atrial pacing compared to the control mice (Fig.11).
[0078] • GIRK4p.G387R+/+ mice displayed heart rate variability at baseline. Genetic inactivation of GIRK4 in mice showed reduced heart rate variability characterized by reduced high frequency (HF) and low frequency (LF) parameters of heart rate variability spectra (HRV). In contrast, GIRK4p.G387R+/+ mice displayed increased HF and LF variability compared to control littermates with no changes in basal heart rate and RR interval ( Fig.12).
[0079] • Left atrial changes are noted in GIRK4p.G387R+/+ mice. Echocardiographic studies denoted a significant increase in left atrial diameter at 12 and 20 wks. of age consistent with AF patients. Mice did not display changes in left ventricular internal diameter in diastole (LVID, d) at both time points. No changes in ejection fraction (EF) were seen at 12 wks. and only a mild reduction in EF was noted later at 20 wks. of age ( Fig.13).
[0080] • GIRK4p.G387R+/+ atrial myocytes displayed aberrant /K Ach fast desensitization. /K ACH was induced by a square voltage pulse stepping from -80 mV to -lOOmV before and after lOuM carbachol (CCh) at 29°C. Desensitization is expressed as ratio of /K.ACH at f=10s over /peak (peak at the beginning) (Fig.14).
[0081] Structural characterization of hiPSC-aCMs. We have developed techniques to culture and differentiate hiPSCs. hiPSCs control line was generated by the Stanford iPSC Biobank. hiPSCs were differentiated into ventricular cardiomyocytes (hiPSC-vCMs) via small moleculebased modulation of Wnt signaling, as described by Burridge et al., Nat Methods. 2014;ll(8):855-60 and Lian et aL, Nat Protoc. 2013;8(l):162-75. Atrial subtype differentiation (hiPSC-aCMs) was performed as described by Cyganek. hiPSC-vCMs generated under the control condition showed robust expression of ventricle-specific myosin light chain 2 (MLC2V) and lower levels of atrial MLC2A. hiPSC-aCMs obtained from the RA-treated cultures revealed high expression of MLC2A, but no expression of MLC2V. In addition, hiPSC-aCMs displayed increased expression of connexin-40 while hiPSC-vCMs displayed an increased expression of connexin-43 (Fig.15).
[0082] Action potential recordings from iPSC-aCMs displayed increased response of atrial myocyte to the modulator (carbachol, CCh). CCh shortened APD. In addition, iPSC-aCMs
Figure imgf000022_0001
showed increased GIRK1 and GIRK4 protein levels compared to iPSC-vCMs. Of note, KCNJ3 was identified to be highly expressed in (iPSC-aCMs) (Fig.16).
[0083] TAT-GIRKl-C-terminus peptide shows reduced /K.ACH compared to ML297 (/K AC activator) and control cells. Mice lacking /K,ACH via genetic inactivation were demonstrated to be resistant to CCh-induced AF. In contrast, CCh administration induced AF in 10 of 14 WT mice. Such findings suggest that inhibition or abolishment of /K.ACH could be preventative against AF, thereby implicating a therapeutic role of KAch inhibitors in the treatment of AF. /K,ACH was recorded in iPSC-aCMs. /K,ACH was characterized as a CCh induced current (Fig.17A). Currents were measured before and after addition of CCh (10uM) in response to a 350ms voltage ramp protocol ranging from 20mV to -120mV with a holding potential of -40mV. /K.AC was quantified by subtraction of the current recorded without CCh from the current recorded with CCh. The currents were normalized to cell capacitance to obtain the current density. /K,ACh current-voltage relationship in control, ML297, and GIRK1 C-terminus peptide showed significant differences. Compared to control group, ML297 significantly increased /K,ACh in negative voltage range from -70mV to - 120mV, whereas the peptide significantly decreased /K,ACh in the range from -90mV to -120mV (Fig.l7B-C). Finally, both peptides reduced normalized GIRK4 membrane expression after treatment in HL-1 cells compared to the control (PBS) as shown in immunoblotting following surface biotinylation (Fig.17D-E).
ADDITIONAL ASPECTS
[0084] Aspect 1: A composition comprising an amino acid sequence, wherein said amino acid sequence comprises about 20-60 residues of SEQ. ID NO: 1, wherein the sequence is conjugated to a cell penetration peptide.
[0085] Aspect 2: The composition of aspect 1, wherein said amino acid sequence comprises residues at or near the C terminus of SEQ. ID NO: 1.
[0086] Aspect 3: The composition of aspect 1 or aspect 2, wherein the amino acid sequence comprises 90% or more identity to SEQ. ID NO: 2.
[0087] Aspect 4: The composition according to any one of aspects 1-3, wherein the cell penetration peptide comprises a cationic cell penetrating peptide, a hydrophobic cell penetrating peptide, or an amphiphatic cell penetrating peptide.
[0088] Aspect 5: The composition according to any one of aspects 1-4, wherein the cell penetrating peptide comprises any one of SEQ. ID NO: 3-92.
[0089] Aspect 6: The composition according to any one of aspects 1-5, wherein the cell penetrating peptide is selected from one of the following sequences:
Seq. ID NO: 3 - GRKKRRQRRRPPQ
Seq. ID NO: 4 - YGRKKRRQRRR
Seq. ID NO: 5 - CYGRKKRRQRRR
Seq. ID NO: 6 - RKKRRQRRR
Seq. ID NO: 7 - TRQARRNRRRRWRERQR
Seq. ID NO: 8 - RQIKIWFQNRRMKWKK
Seq. ID NO: 9 - LLIILRRRIRKQAHAHSK
Seq. ID NO: 10 - MVTVLFRRLRIRRACGP- PRVRV Seq. ID NO: 11 - MVRRFLVTLRIRRACG- PPRVRV Seq. ID NO: 12 - INLKALAALAKKIL Seq. ID NO 13 - AGYLLGKINLKALAAL- AKKIL
Seq. ID NO 14 - CGYGPKKKRKVGG
Seq. ID NO 15 - PKKKRKV
Seq. ID NO 16 - KWRRKLKKLRPKKKR- KV
Seq. ID NO 17 - KWRRKLKKLR
Seq. ID NO 18 - LGTYTQDFNKFHTFPQT- AIGVGAP
Seq. ID NO 19 - RKKRRRESRKKRRRES
Seq. ID NO 20 - HSDGTFTSELSRLRDSA- RLQRLLQGLV
Seq. ID NO 21 - LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
Seq. ID NO 22 - SQPEATKCFQWQRNM RKVRGPPVSCIKRDSPIQI
Seq. ID NO 23 - GRGDSY
Seq. ID NO 24 - (VRLPPP)3
Seq. ID NO 25 - KCFQWQRNMRKVRGP- PVSCIKR
Seq. ID NO 26 - RRIRPRPPRLPRPRPRPL- PFPRPG
Seq. ID NO 27 - RAGLQFPVG[RLLR]3
Seq. ID NO 28 - GLRKRLRKFRNKIKEK
Seq. ID NO 29 - RGGRLCYCRRRFCVCVG-R
Seq. ID NO 30 - MVKSKIGSWILVLFVAMWSDVGLCKKRP
Seq. ID NO 31 - DPRSFL
Seq. ID NO 32 - NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ
Seq. ID NO 33 - DAATATRGRSAASRPTERPRAPARSASRPRRPVE
Seq. ID NO 34 - DPKGDPKGVTVTVTVT- VTGKGDPKPD
Seq. ID NO 35 - PIEVCMYREP
Seq. ID NO 36 - CSIPPEVKFNKPFVYLI
Seq. ID NO 37 - LSTAADMQGVVTDGMASGLDKDYLKPDD
Seq. ID NO 38 - PFVYLI
Seq. ID NO 39 - RLSGMNEVLSFRWL
Seq. ID NO 40 - SDLWEMM MVSLACQY
Seq. ID NO 41 - CRWRWKCCKK
Seq. ID NO 42 - WLRRIKAWLRRIKALN- RQLGVAA
Seq. ID NO 43 - GLFGAIAGFIENGWEGM- IDGWYG
Seq. ID NO 44 - WEAKLAKALAKALAHLAKALAKALKACEA
Seq. ID NO 45 - LSTAADMQGWTDGMASGLDKDYLKPDD
Seq. ID NO 46 - PIEVCMYREP
Seq. ID NO 47 - VPTLK
Seq. ID NO 48 - RRQRRTSKLMKR
Seq. ID NO 49 - KGRKKRRQRRRPPQ
Seq. ID NO 50 - RRRRNRTRRNRRRVR-amide
Seq. ID NO 51 - Acetyl-KLALKLALKALKAALKL-A-amide
Seq. ID NO 52 - PLSSIFSRIGDP
Seq. ID NO 53 - RPKPQQFGLM-amide
Seq. ID NO 54 - YKQSHKKGGKKGSG
Seq. ID NO 55 - RRRRRRRRR
Seq. ID NO 56 - GLFKALLKLLKSLWKL- LLKA
Seq. ID NO 57 - WEAKLAKALAKALAKHLAKALAKALKACEA
Seq. ID NO 58 - RQIRIWFQNRRMRWRR
Seq. ID NO 59 - RRRRRRHHHH
Seq. ID NO 60 - GLWRALWRLLRSLWR- LLWRA Seq. ID NO 61 - KAFAKLAARLYRKALA- RQLGVAA Seq. ID NO 62 - KETWWETWWTEWSQP- KKKRKVCya Seq. ID NO 63 - GLFRALLRLLRSLWRLL-LRA Seq. ID NO 64 - RAGLQFPVGRLLRRLLR Seq. ID NO 65 - RRRR Seq. ID NO 66 - RRRRRR Seq. ID NO 67 - RRRRRRRRRR Seq. ID NO 68 - RRRRRRRRRRRR Seq. ID NO 69 - GALFLGWLGAAGSTM- GAPKKKRKV Seq. ID NO 70 - CHHHHHRRRRRRRR- RHHHHHC Seq. ID NO 71 - ac-KWFETWFTEWPKKR- K-Cya Seq. ID NO 72 - KKKK Seq. ID NO 73 - ALFLGFLGAAGSTMGA- WSQPKKKRKV Seq. ID NO 74 - RRRRRRRR Seq. ID NO 75 - KKKKKKKK Seq. ID NO 76 - KKKKKK Seq. ID NO 77 - KKKKKKKKKK Seq. ID NO 78 - KKKKKKKKKKKK Seq. ID NO 79 - RRQRR Seq. ID NO 80 - RRQRRQRR Seq. ID NO 81 - RRQRRQRRQRR Seq. ID NO 82 - GALFLGFLGAAGSTMGAWSQPKSKRKV Seq. ID NO 83 - RRRRRRRRRRRRRRR Seq. ID NO 84 - HHHHHHHHRRRRRR- RRRRRRRRR Seq. ID NO 85 - HHHHHHHHHHHHHHHHRRRRRRRRRRRRRRR Seq. ID NO 86 - ISFDELLDYYGESGS Seq. ID NO 87 - GYGYGYGYGYGYGYGYKKRKKRKKRKKRKQ-QKQQKRRK Seq. ID NO 88 - KWLLRWLSRLLRWLAR WLG Seq. ID NO 89 - LLWRLWRLLWRLWRLL Seq. ID NO 90 - KLALKLALKALKAALK- LA Seq. ID NO 91 - ALWKTLLKKVLKAPKK-KRKVC Seq. ID NO 92 - GWTLNSAGYLLGKINL- KALAALAKKIL
[0090] Aspect 7: The composition according to any one of aspects 1-6, wherein the amino acid sequence is conjugated to the cell penetrating peptide through a linker.
[0091] Aspect 8: The composition according to any one of aspects 1-7 wherein the amino acid sequence is conjugated to the cell penetrating peptide through a linker having the sequence - (G)xS-x, wherein x is from 3 to 10.
[0092] Aspect 9: A composition comprising an amino acid sequence, wherein said amino acid sequence comprises 20-60 residues of SEQ. ID NO: 1.
[0093] Aspect 10: The composition of aspect 9, wherein said amino acid sequence comprises residues at or near the C terminus of SEQ. ID NO: 1. [0094] Aspect 11: The composition of aspect 9 or aspect 10, wherein the amino acid sequence comprises 90% or more, 95% of more, or 98% or more identity to SEQ. ID NO: 2.
[0095] Aspect 12: A composition comprising an amino acid sequence having the formula: GRKKRRQRRRPPQ-GGGGS- GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR.
[0096] Aspect 13: A composition comprising an amino acid sequence having the formula: PIEVCMYREP-GGGGS-GREDFPKKLLRMSSTTSEKAYSLGDLPMKLQR
[0097] Aspect 14: The composition of any of aspects 1-13, wherein the composition is a lyophilized powder and optionally one or more pharmaceutically acceptable excipients.
[0098] Aspect 15: The composition of any of aspects 1-13, wherein the composition comprises water and optionally one or more pharmaceutically acceptable excipients.
[0099] Aspect 16: A method of regulating lK,Ach activity and reducing AF inducibility, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0100] Aspect 17: A method of downregulating GIRK4 membrane expression, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0101] Aspect 18: A method of reducing the formation of GIRK tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0102] Aspect 19: A method of reducing the formation of abnormal GIRK4 homo-tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0103] Aspect 10: A method of treating a disorder characterized by aberrant lK,Ach activity and arrhythmic activity, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0104] Aspect 21: A method of treating a disorder characterized by upregulation of membrane GIRK4, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0105] Aspect 22: A method of treating a disorder characterized by excess formation of GIRK4 homo-tetramers, comprising administering to a subject in need thereof the composition according to any one of aspects 1-15.
[0106] Aspect 23: The method of any one of aspects 16-22, wherein the subject has a disorder characterized by an arrhythmia or structural heart disease leading to arrhythmia.
[0107] Aspect 24: The method of aspect 23, wherein the arrhythmia is tachycardia, bradycardia, or a combination thereof. [0108] Aspect 25: The method of any one of aspects 23 or 24, wherein the arrhythmia is atrial fibrillation, atrial flutter, supraventricular tachycardia, sick sinus syndrome, atrioventricular dysfunction, tachy-brady syndrome, or a combination thereof.
[0109] Aspect 26: The method of any one of aspects 23-25, further administering to the subject a beta blocker, calcium channel blocker, sodium channel blocker, potassium channel blocker, or combination thereof.
[0110] Aspect 27: The method of any one of aspects 23-26, further administering to the subject atenolol, bisoprolol, carvedilol, metoprolol, propranolol, timolol, diltiazem, verapamil, digoxin, flecainide, propafenone, quinidine, amiodarone, sotalol, dofetilide, dronedarone, or a combination thereof.
[0111] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

Claims

CLAIMS What is claimed is:
1. A composition comprising a therapeutic peptide having an amino acid sequence, wherein the therapeutic peptide comprises no more than 200 amino acid residues, and the amino acid sequence comprises a sequence of at least 20 amino acids having 90% or greater similarity to a 20 amino acid sequence from [Seq. ID NO: 1],
2. The composition according to claim 1, wherein the 20 amino acid sequence from [Seq. ID NO: 1] is located within 100 amino acid residues from the C-terminus of [Seq. ID NO: 1],
3. The composition according to claim 1, wherein the amino acid sequence has 90% or greater similarity to [Seq. ID NO: 2].
4. The composition according to claim 1, wherein the amino acid sequence has 90% or greater similarity to [Seq. ID NO: 93],
5. The composition according to claim 1, wherein the amino acid sequence has 90% or greater similarity to [Seq. ID NO: 94],
6. The composition according to claim 5, wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
7. The composition according to claim 5, wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
8. The composition according to claim 1, wherein the amino acid sequence has 90% or greater similarity to [Seq. ID NO: 95],
9. The composition according to claim 6, wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
10. The composition according to claim 6, wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
11. The composition according to any of claims 1-10, wherein the therapeutic peptide further comprises a cell penetrating peptide.
12. The composition according to claim 11, wherein the cell penetration peptide comprises a cationic cell penetrating peptide, a hydrophobic cell penetrating peptide, or an amphiphatic cell penetrating peptide.
13. The composition according to claim 11, wherein the cell penetrating peptide comprises any one of Seq. ID NO: 3-92.
14. The composition according to claim 11, wherein the cell penetrating peptide is covalently conjugated to the therapeutic peptide through an amide bond or a linker.
15. The composition according to claim 14, wherein the linker comprises any one of Seq. ID NO: 96-99.
16. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to any one of Seq. ID NO: 100-107.
17. The composition according to claim 16, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 100.
18. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 101.
19. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 102.
20. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 103.
21. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 104.
22. The composition according to claim 21, wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
23. The composition according to claim 21, wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
24. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 105.
25. The composition according to claim 24, wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
26. The composition according to claim 24, wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
27. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 106.
28. The composition according to claim l , wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
29. The composition according to claim l , wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
30. The composition according to claim 1, wherein the therapeutic peptide has 90% or greater similarity to Seq. ID NO: 107.
31. The composition according to claim 30, wherein X1 is K and X2 is N, Q, K, D, E, I, Y, or W.
32. The composition according to claim 30, wherein X1 is E, R, Q, S, Y, or W, and X2 is S.
33. The composition according to any of claims 1-33, comprising a solvent.
34. The composition according to any of claims 1-34, comprising an aqueous carrier.
35. The composition of any of claims 1-33, wherein the composition is a lyophilized powder.
36. A method of regulating lK,Ach activity and reducing AF inducibility, comprising administering to a subject in need thereof the composition according to any of claims 1-35.
37. A method treating a disorder characterized by aberrant lK,Ach activity and arrhythmic activity, comprising administering to a subject in need thereof the composition according to any of claims 1-35.
38. A method of treating arrhythmia in a subject in need thereof, comprising administering to a subject in need thereof the composition according to any of claims 1-35.
39. The method according to claim 38, wherein the arrhythmia comprises tachycardia, bradycardia, or a combination thereof.
40. The method according to claim 38, wherein the arrhythmia comprises atrial fibrillation, atrial flutter, supraventricular tachycardia, sick sinus syndrome, atrioventricular dysfunction, tachy-brady syndrome, or a combination thereof.
41. The method according to any of claims 37-40, further comprising administering to the subject a beta blocker, calcium channel blocker, sodium channel blocker, potassium channel blocker, or combination thereof.
42. The method according to any of claims 37-40, further comprising administeringatenolol, bisoprolol, carvedilol, metoprolol, propranolol, timolol, diltiazem, verapamil, digoxin, flecainide, propafenone, quinidine, amiodarone, sotalol, dofetilide, dronedarone, or a combination thereof.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050106644A1 (en) * 2001-06-20 2005-05-19 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
CN101343327B (en) * 2008-08-13 2012-05-23 汕头大学医学院 Antiviral protein and application thereof
US20230054711A1 (en) * 2019-12-31 2023-02-23 Xiamen University Multimerization delivery system for intracellular delivery of molecule

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050106644A1 (en) * 2001-06-20 2005-05-19 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
CN101343327B (en) * 2008-08-13 2012-05-23 汕头大学医学院 Antiviral protein and application thereof
US20230054711A1 (en) * 2019-12-31 2023-02-23 Xiamen University Multimerization delivery system for intracellular delivery of molecule

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