Polypeptide conjugate and use thereof in heart failure with preserved ejection fraction

Abstract

Provided is a polypeptide conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide conjugate comprises: (a) a degradation-inducing ligand, (b) a cyclic peptide, and (c) a Jun-targeting protein peptide. The polypeptide conjugate can be used for treating heart failure with preserved ejection fraction.

Description

Peptide conjugates and their application in heart failure with preserved ejection fraction Technical Field

[0001] The present invention relates to the field of disease treatment, and in particular provides a polypeptide conjugate for degrading Jun protein and its application in heart failure with preserved ejection fraction. Background Art

[0002] Cardiovascular disease is one of the leading causes of death worldwide, bringing a heavy medical and economic burden. Heart failure severely limits people's quality of life. Heart failure includes systolic heart failure and diastolic heart failure. With the development of medicine, diastolic heart failure has also been more broadly defined as heart failure with preserved ejection fraction (HFpEF). HFpEF is a syndrome with high morbidity and mortality. According to statistics, the global mortality rate due to HF is 35%, of which HFpEF accounts for more than 50%.

[0003] To date, there is a lack of effective drug therapies proven to modify disease progression and prognosis in patients with HFpEF, resulting in a huge unmet clinical need. Summary of the Invention

[0004] Our research group previously discovered through single-cell sequencing that the target JUN is closely associated with heart failure (HFpEF). Overexpression of JUN leads to HFpEF, and intervention in JUN can ameliorate the disease progression. However, drug development targeting JUN currently faces numerous challenges. Traditional drug design approaches primarily target target proteins by developing various small molecule, peptide, and protein inhibitors. These inhibitors occupy and block the target protein's active sites, inhibiting its functional activity and thus exerting therapeutic efficacy. However, these traditional development strategies are limited by their mechanisms of action and present the following challenges: 1) Most target proteins, such as kinases and ion channels, contain druggable active pockets or binding sites. This makes it difficult to identify high-affinity inhibitors for many potential drug targets without well-defined active sites, such as scaffolding proteins, transcription factors, and non-enzyme proteins. 2) Inhibitors bind specifically to the target protein's active site in an "occupancy-driven" manner, relying on maintaining high drug concentrations for a sufficient period of time to fully occupy the target protein's binding site. However, high drug concentrations can easily lead to side effects such as off-target effects and adverse reactions. 3) Small molecule inhibitors may induce compensatory protein activity or genetic mutations, leading to drug resistance. Considering the limitations of inhibitors, researchers have developed technologies to regulate protein expression at the genetic level, such as RNA interference (RNA interference) and DNA-based CRISPR / Cas9. Although gene interference technology overcomes the limitations of protein structure on drug design, it faces challenges such as irreversible effects, a high risk of off-target effects, and prolonged effects that can easily trigger compensatory mechanisms in cells, severely restricting its development and therapeutic potential. Therefore, developing a method for targeted degradation of specific proteins would overcome the many bottlenecks of existing drug development technologies and provide new avenues for new drug development.

[0005] Peptide conjugates

[0006] In one aspect, the present invention provides a polypeptide conjugate or a pharmaceutically acceptable salt thereof, comprising: (a) a degradation-inducing ligand, (b) a cyclic peptide, and (c) a Jun protein targeting peptide; wherein the Jun protein targeting peptide is selected from QLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 24) or EEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 25).

[0007] In certain embodiments, the polypeptide conjugate comprises the degradation-inducing ligand, the cyclic peptide, and the Jun protein targeting peptide from the N-terminus to the C-terminus. In certain embodiments, the polypeptide conjugate consists of the degradation-inducing ligand, the cyclic peptide, and the Jun protein targeting peptide from the N-terminus to the C-terminus, and the degradation-inducing ligand, the cyclic peptide, and the Jun protein targeting peptide are optionally connected to each other by a linker.

[0008] In certain embodiments, the cyclic peptide has a structure shown in Formula I:

[0009] Cyclic [isoD-X1-X2-Dap](I)

[0010] Wherein, isoD is isoaspartic acid, Dap is 2,3-diaminopropionic acid (2,3-Diaminopropionic acid), X1 and X2 are each independently selected from natural amino acids, and the isoD and Dap form a cyclization.

[0011] In certain embodiments, X1 and X2 are each independently selected from L-amino acids.

[0012] In certain embodiments, X1 is E or V. In certain embodiments, X2 is I or L.

[0013] In certain embodiments, the cyclic peptide is Cyclo(isoD-EI-Dap) or Cyclo(isoD-VL-Dap).

[0014] In certain embodiments, the isoD and Dap are covalently linked via their respective side chains. In certain embodiments, the isoD is linked via an amide bond via the carboxyl group of the side chain and the amino group of the side chain of Dap.

[0015] In certain exemplary embodiments, the cyclic peptide has The structure shown.

[0016] In certain embodiments, the cyclic peptide is Cyclo(isoD-EI-Dap), and the Jun protein targeting peptide is QLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 24).

[0017] In certain embodiments, the cyclic peptide is Cyclo(isoD-VL-Dap), and the Jun protein targeting peptide is EEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 25).

[0018] In certain embodiments, the degradation-inducing ligand refers to a ligand responsible for targeting the ubiquitin-proteasome system (UPS) or the autophagy-lysosome pathway (ALP) in targeted protein degradation (TPD) technology to induce protein degradation.

[0019] In certain embodiments, the degradation-inducing ligand is a ligand in a targeted protein degradation technology based on the ubiquitin-proteasome system (UPS). In certain embodiments, the targeted protein degradation technology based on the ubiquitin-proteasome system (UPS) is a proteolysis targeting chimaera (PROTAC), which is a class of heterobifunctional molecules formed by connecting a target protein ligand and an E3 ligase ligand through an appropriate linker. It recruits the E3 ligase to the vicinity of the target protein to form a ternary complex, thereby inducing ubiquitination and proteasomal degradation of the target protein.

[0020] In certain embodiments, the degradation-inducing ligand is an E3 ubiquitin ligase ligand, such as LAP(OH)YI(Leu-Ala-Pro OH -Tyr-Ile, SEQ ID NO: 26).

[0021] In certain embodiments, the degradation-inducing ligand is a ligand in a targeted protein degradation technology based on the autophagy-lysosomal pathway (ALP). In certain embodiments, the targeted protein degradation technology based on the autophagy-lysosomal pathway (ALP) is an autophagosome-tethering compound (ATTEC), in which an LC3 ligand is connected to a different target protein ligand via a linker to form a heterobifunctional autophagy degrader. Under the regulation of cellular autophagy-inducing signals, microtubule-associated protein 1A / 1B light chain 3 (LC3) is lipidated from cytoplasmic LC3 (LC3-I) to autophagosomal membrane LC3 (LC3-II), which is adsorbed on the phagophore, a cup-shaped double-membrane structure formed by the free membranes of organelles such as the Golgi apparatus and the endoplasmic reticulum, and further extends; the phagophore further wraps around target proteins and other substrates in the cytoplasm to form a closed compartment with a double-membrane structure, namely the autophagosome; finally, the autophagosome fuses with the lysosome to form an autolysosome for subsequent degradation.

[0022] In certain embodiments, the degradation-inducing ligand is an LC3 ligand, such as WGPIWGPI (SEQ ID NO: 27).

[0023] In certain embodiments, (a), (b), and (c) are covalently linked to each other.

[0024] In certain embodiments, (a), (b), and (c) are optionally connected to each other via a linker or directly (eg, via a peptide bond).

[0025] In certain embodiments, the linker is a spacer group. In certain embodiments, the linker is selected from aminocaproic acid (Ahx) or 12-aminolauric acid (12-Ado).

[0026] In some embodiments, (a) and (b) are directly connected. In some embodiments, (a) and (b) are connected via a linker.

[0027] In certain embodiments, (b) and (c) are directly connected.

[0028] In certain embodiments, (a) and (b) are directly connected or connected through a linker, and (b) and (c) are directly connected.

[0029] In certain embodiments, the degradation-inducing ligand is LAP(OH)YI (SEQ ID NO: 26) or WGPIWGPI (SEQ ID NO: 27), the cyclic peptide is Cyclo (isoD-EI-Dap), and the peptide targeting Jun protein is QLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 24).

[0030] In certain embodiments, the degradation-inducing ligand is WGPIWGPI (SEQ ID NO: 27), the cyclic peptide is Cyclo (isoD-VL-Dap), and the peptide targeting Jun protein is EEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 25).

[0031] In certain embodiments, the polypeptide conjugate has a structure selected from the group consisting of:

[0032] WGPIWGPI-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:16);

[0033] WGPIWGPI-ahx-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:17);

[0034] WGPIWGPI-12Ado-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:18);

[0035] LAP(OH)YI-ahx-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:19);

[0036] LAP(OH)YI-12Ado-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 20).

[0037] In certain embodiments, the polypeptide conjugate has a structure selected from the group consisting of:

[0038] WGPIWGPI-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:21);

[0039] WGPIWGPI-ahx-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:22);

[0040] WGPIWGPI-12Ado-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 23).

[0041] In certain embodiments, the polypeptide conjugate has a structure selected from the group consisting of:

[0042] LAP(OH)YI-ahx-Cyclo(isoDVLDap)EEIEQLEERNYALRKEIEDLQKQLEKL(SEQ ID NO:31);

[0043] LAP(OH)YI-12Ado-Cyclo(isoDVLDap)EEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 32).

[0044] In certain embodiments, the polypeptide conjugate described in any of the above embodiments further comprises a nuclear localization sequence. The term "nuclear localization sequence" or "NLS" is used to refer to an amino acid sequence that marks a protein so that it can be imported into the cell nucleus through nuclear transport. Typically, the nuclear localization sequence consists of a short peptide rich in positively charged amino acids (e.g., lysine and / or arginine). In certain embodiments, the nuclear localization sequence is located at the C-terminus of the polypeptide conjugate. In certain embodiments, the nuclear localization sequence is derived from the SV40 virus T antigen. In certain embodiments, the nuclear localization sequence is PKKKRKV (SEQ ID NO: 28).

[0045] In certain embodiments, the polypeptide conjugate carrying a nuclear localization sequence has a structure selected from the group consisting of:

[0046] WGPIWGPI-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:5);

[0047] WGPIWGPI-ahx-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:6);

[0048] WGPIWGPI-12Ado-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:7);

[0049] LAP(OH)YI-ahx-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:8);

[0050] LAP(OH)YI-12Ado-Cyclo(isoD-EI-Dap)QLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:9);

[0051] WGPIWGPI-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:11);

[0052] WGPIWGPI-ahx-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:12);

[0053] WGPIWGPI-12Ado-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:13);

[0054] LAP(OH)YI-ahx-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKLPKKKRKV(SEQ ID NO:14);

[0055] LAP(OH)YI-12Ado-Cyclo(isoD-VL-Dap)EEIEQLEERNYALRKEIEDLQKQLEKLPKKKRKV (SEQ ID NO: 15).

[0056] In certain embodiments, the polypeptide conjugates described in any of the above embodiments comprise an N-terminal modification and / or a C-terminal modification, such as an N-terminal modification using suitable amino-reactive chemistry and / or a C-terminal modification using suitable carboxy-reactive chemistry. In certain embodiments, the N-terminal modification or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a detectable label.

[0057] In certain embodiments, the polypeptide conjugate described in any of the above embodiments comprises an N-terminal capping group, such as a formyl group, an acetyl group, or a benzoyl group. In certain embodiments, the polypeptide conjugate comprises an N-terminal acetyl group. In such embodiments, the N-terminal residue is capped with acetic anhydride or other appropriate reagent during peptide synthesis to produce an N-terminally acetylated molecule.

[0058] In certain embodiments, the polypeptide conjugate described in any of the above embodiments comprises a C-terminal modification. In certain embodiments, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule.

[0059] In certain embodiments, the polypeptide conjugate described in any of the above embodiments comprises a detectable label. In certain embodiments, the polypeptide conjugate comprises a detectable label at its N-terminus. The detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemistry. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes, chemiluminescent substances (such as acridinium ester compounds) or biotin. In certain embodiments, the polypeptide conjugate comprises biotin at its N-terminus.

[0060] Pharmaceutically acceptable salts

[0061] It will be understood that salt forms are within the scope of the present invention and reference to a polypeptide conjugate includes salt forms of the polypeptide conjugate.

[0062] The pharmaceutically acceptable salt of the polypeptide conjugate of the present invention may be in any suitable pharmaceutically acceptable salt form. The term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the polypeptide conjugates provided by the present invention and a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. and (ii) salts formed between basic functional groups (e.g., -NH2) present in the polypeptide conjugates provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, and hydroiodides; inorganic acid salts, such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; arylsulfonates, such as benzenesulfonates and p-toluenesulfonates; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, and aspartate.

[0063] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods, for example, as described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or an organic solvent, or in a mixture of the two.

[0064] Pharmaceutical compositions and therapeutic applications

[0065] In another aspect, the present invention provides a pharmaceutical composition comprising the polypeptide conjugate of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0066] In another aspect, the present invention provides use of the polypeptide conjugate of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for preventing and / or treating a disease. Also provided are methods for preventing and / or treating a disease in a subject, comprising administering an effective amount of the polypeptide conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a subject in need thereof.

[0067] In certain embodiments, the disease is a Jun-related disease.

[0068] In certain embodiments, the disease is characterized by overexpression of Jun or excessive Jun activity.

[0069] In certain embodiments, the Jun-related disease involves Jun overexpression. Jun overexpression refers to Jun levels (eg, Jun levels in the subject's plasma or tissue, preferably myocardial tissue) that are higher than normal Jun levels (eg, corresponding levels in healthy controls).

[0070] In certain embodiments, the Jun-associated disease would benefit from reduced levels of Jun or inhibition of expression.

[0071] In certain embodiments, the polypeptide conjugate of the present invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, prevents or treats the disease by degrading Jun protein.

[0072] In certain embodiments, the disease is heart failure with preserved ejection fraction (HFpEF).

[0073] In certain embodiments, the subject is a human.

[0074] The polypeptide conjugate or pharmaceutically acceptable salt or pharmaceutical composition of the present invention can be used alone or in combination with another pharmaceutically active agent.

[0075] The polypeptide conjugates of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The polypeptide conjugates of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by incorporating the necessary dose of the active ingredient into an appropriate solvent, and optionally, other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Additionally, sterile injectable solutions can be prepared as sterile lyophilized powders (eg, by vacuum drying or freeze drying) for ease of storage and use.

[0076] The polypeptide conjugates of the present invention or their pharmaceutically acceptable salts or pharmaceutical compositions can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the polypeptide conjugates of the present invention or their pharmaceutically acceptable salts or pharmaceutical compositions are administered by intravenous or bolus injection.

[0077] The polypeptide conjugates of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

[0078] Definition of terms

[0079] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0080] As used herein, the term "heart failure with preserved ejection fraction (HFpEF)" refers to a clinical syndrome in which, in the presence of normal or mildly reduced ventricular systolic function, impaired ventricular diastolic function and reduced compliance lead to reduced ventricular filling and increased filling pressure, thereby causing congestion in the pulmonary and systemic circulations. HFpEF typically refers to heart failure with diastolic dysfunction. In certain embodiments, the clinical diagnostic criteria for HFpEF mainly include: (1) the presence of symptoms and / or signs of heart failure; (2) cardiac imaging examination (mainly TTE examination) indicating LVEF ≥ 50%; (3) objective evidence of cardiac structural and / or functional abnormalities consistent with left ventricular diastolic dysfunction and / or increased left ventricular filling pressure, wherein the structural and / or functional abnormality indicators of left ventricular diastolic dysfunction and / or increased ventricular filling pressure mainly include: (a) mean E / e' ratio > 15; (b) left atrial volume index > 40 ml / m 2 (Atrial Fibrillation).

[0081] As used herein, the term "Jun" refers to the Jun proto-oncogene, AP-1 transcription factor subunit (Jun proto-oncogene, AP-1 transcription factor subunit), also known as AP1, AP-1, cJUN, or c-Jun. Jun can be human or a homologous gene from another species (e.g., non-human mammals, fish, reptiles, or birds, such as rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, etc.). The sequence of Jun is well known to those skilled in the art and can be found in various public databases. An exemplary gene sequence of human Jun can be found in GenBank: NM_002228.4, and an exemplary protein sequence can be found in NCBI: NP_002219.1; an exemplary gene sequence of mouse Jun can be found in Ensembl: ENSMUSG00000052684, NCBI Gene ID: 16476, and an exemplary protein sequence can be found in UniProtKB: P05627, NCBI: NP_034721.1.

[0082] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin.

[0083] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (if not receiving treatment).

[0084] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with factors such as the patient's disease state, age, sex, body weight, etc. An effective amount is also the amount at which the therapeutic beneficial effect exceeds any toxic or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following results, such as eliminating or reducing risk, alleviating severity, or delaying the onset of the disease, including the biochemistry of the disease, histology and / or behavioral symptoms, the intermediate pathological phenotype presented during its complications and disease formation. For therapeutic use, the beneficial or desired result includes clinical results, such as reducing one or more symptoms derived from the disease, improving the quality of life of those subjects suffering from the disease, reducing the dosage of other drugs needed for the treatment of the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and / or prolonging survival. An effective amount can be used in one or more administrations. Beneficial effects

[0085] The inventors of the present application have provided for the first time a polypeptide conjugate that can inhibit the occurrence and development of HFpEF and improve diastolic function, which has important clinical value for the prevention and treatment of HFpEF.

[0086] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1: Construction of the HFpEF model. A: Schematic diagram of the experimental process; B: Diastolic function (E / A) of mice after 5 weeks of feeding; C: Diastolic function (E / E') of mice after 5 weeks of feeding.

[0088] Figure 2: Jun expression in HFpEF mouse model.

[0089] Figure 3A-Figure 17A: HPLC profiles of polypeptide conjugates.

[0090] Figure 3B-Figure 17B: MS spectra of polypeptide conjugates.

[0091] Figure 18-Figure 19: Evaluation of degradation activity of polypeptide conjugates at the cellular level.

[0092] Figure 20: In vivo evaluation of the efficacy of peptide J7 in a HFpEF mouse model. A: Schematic diagram of the experimental process; B: Diastolic function (E / E') test results.

[0093] Sequence information

[0094] A description of the sequences involved in this application is provided in the table below.

[0095] Table 1: Sequence information Example

[0096] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0097] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0098] Example 1: Jun expression is correlated with HFpEF

[0099] Experimental materials and methods

[0100] Animals: C57BL / 6N wild-type mice were purchased from Beijing Weitonglihua;

[0101] The reagents are as follows:

[0102] Table 2: Reagent information

[0103] Animal Experimentation Guidelines

[0104] In this example, all animal studies were conducted under the guidance of the Laboratory Animal Center, Fuwai Hospital Animal Care and Use Committee, National Center for Cardiovascular Diseases, China. All mice were propagated and housed under the same conditions and randomly assigned to groups during the experiment. Echocardiographic analysis was performed by an independent investigator who was unaware of the study objectives.

[0105] Conventional ultrasonic testing

[0106] All mice were fed under different conditions for five weeks and then underwent routine ultrasound examinations every two weeks until the end of the fifteen-week monitoring period. Specifically, transthoracic echocardiography was performed using a VisualSonics Vevo 2100 system equipped with an MS400 transducer (Visual Sonics). Left ventricular ejection fraction (LVEF) and other systolic function indicators were obtained from short-axis M-mode scans at the level of the mid-ventricle, as indicated by the presence of papillary muscles, in conscious, lightly restrained mice. Apical four-chamber views were obtained in anesthetized mice for diastolic function measurements using pulsed wave and tissue Doppler imaging at the level of the mitral valve. Anesthesia was induced by 2.5% isoflurane and confirmed by the lack of response to firm pressure on one of the hind paws. Isoflurane was reduced to 1.0-1.5% during echocardiographic acquisition (under temperature-controlled conditions) and adjusted to keep the heart rate within 500 beats per minute. Parameters collected included heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, end-diastolic interventricular septal wall thickness, left ventricular end-diastolic posterior wall thickness, left ventricular fractional shortening, left ventricular ejection fraction (LVEF), peak Doppler velocity across the mitral valve in early diastole, peak Doppler velocity across the mitral valve in late diastole, isovolumetric relaxation time, and tissue Doppler peak relaxation velocity at the mitral annulus during early diastole and early filling deceleration. At the end of the procedure, all mice recovered from anesthesia without any abnormalities. All parameters were measured at least three times, and the mean values ​​are presented. Ultrasound testing included both systolic and diastolic function.

[0107] Induction of a heart failure model with preserved ejection fraction

[0108] Eight- to ten-week-old male C57BL / 6N wild-type mice were divided into three groups: a normal group (normal diet and water) and a model group (high-fat diet combined with N-nitro-L-arginine methyl ester). The model group was established using the method described in the following literature: Gabriele G. Schiattarella et al., Nitrosative stress drives heart failure with preserved ejection fraction, https: / / doi.org / 10.1038 / s41586-019-1100-z. Specifically, a high-fat diet (HFD) (60% kcal from fat (lard)) and N-nitro-L-arginine methyl ester (L-NAME, 0.5 g / L in drinking water) were used to induce heart failure with preserved ejection fraction, creating an HFpEF animal model.

[0109] The systolic function parameter LVEF of the mice tested in the fifth week of model induction did not change, while the diastolic function parameter (E / E') increased significantly in the fifth week of model induction, indicating that the heart failure model with preserved ejection fraction described in the aforementioned literature has been successfully obtained. At the same time, there was no significant difference in the diastolic function parameter (E / E') between the model control group and the model treatment group at five weeks, and subsequent drug administration was carried out under the same baseline conditions, as shown in Figure 1.

[0110] Correlation between Jun expression and HFpEF

[0111] At 15 weeks of model induction, myocardial cells from normal mice and the HFpEF model were extracted and separated using a perfusion method, and quantitative RCR detection was performed. The specific procedures are as follows:

[0112] 1. Isolation of adult mouse cardiomyocytes:

[0113] In order to isolate cardiomyocytes from the heart of adult mice, we used the classic perfusion method to isolate cardiomyocytes. Specifically, 100 μl of sodium heparin (1000 units in 50 ml) was injected into the mouse 20 minutes before being killed to prevent heart coagulation during the operation, which increased the difficulty of digestion. After that, the mouse was anesthetized and killed, the heart was removed and transferred to a calcium-free solution for washing. Then, the Langendorff method was used for digestion. The heart was perfused with calcium-free solution for 5 minutes using a Langendorff apparatus, and then digested with a digestive enzyme solution (0.7 mg / ml type II collagenase and 0.7 mg / ml bovine serum albumin calcium-free solution) for about 30 minutes. After about 20 minutes, the heart was constantly touched. When the heart became soft and slippery, it indicated that the digestion was basically completed. Then, the tissue from the ventricle was collected, chopped, and gently blown to dissociate into single cells. The cells were allowed to settle, the supernatant was taken, and the undigested and adherent tissues were removed. 100 g Centrifuge at 4°C for 2 minutes to obtain a myocardial cell pellet. The supernatant is mostly non-myocardial cells. Resuspend the myocardial cells in calcium-free solution containing 10% FBS for subsequent experiments. Non-myocardial cells can be re-selected with culture medium or PBS for subsequent experiments. To obtain purer myocardial cells and non-myocardial cells, centrifuge the cell suspension (100g, 2 minutes at room temperature) three times to separate myocardial cells from non-myocardial cells. Collect myocardial cells for further experiments.

[0114] 2. Quantitative PCR detection:

[0115] Total RNA was extracted from cells using a GeneJet RNA purification kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed using an iScript™ cDNA synthesis kit (Bio-Rad, 1708890) to generate cDNA. qPCR (primer F: SEQ ID NO: 29; primer R: SEQ ID NO: 30) was performed using iTaq Universal SYBR Green supermix (1725121, Bio-Rad) on an ABI Vii7 Real-Time System (Life Technologies, Q6). β-Actin was used for standardized quantitative analysis. As shown in Figure 2, significantly higher expression of Jun was observed in the HFpEF mouse model compared to normal mice. This suggests that Jun expression in mice is correlated with HFpEF and that Jun is highly expressed in HFpEF.

[0116] Example 2: Preparation of polypeptide conjugates

[0117] Wuxi Yapeptide Biotechnology Co., Ltd. was commissioned to synthesize the peptide conjugates described in Table 3, with biotin attached to the N-terminus of each peptide conjugate. Peptide synthesis was performed using standard Fmoc solid-phase synthesis. Rink Amide resin was used, and the peptide chain was elongated from the C-terminus to the N-terminus through deprotection and reaction. The cyclic peptides were removed from the Alloc and Allyl protecting groups using tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid. The ring-closure reaction was then carried out in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole, and N-methylmorpholine, resulting in the formation of a side chain amide ring. Place an appropriate amount of resin in an EP tube, add 1-2 mL of TFA / TIPS / H2O / EDT shearing solution and shake for 1-2 hours. The volume ratio of TFA, TIPS, H2O, and EDT is 94:1:2.5:2.5. Blow the shearing solution dry with nitrogen, then add ether to precipitate for two minutes, centrifuge and discard the supernatant. The precipitated polypeptide is allowed to evaporate the ether in the air. J1, J4-J7, H1, H3, and H6 were purified by high-performance liquid chromatography (HPLC) using a Gemini 5μm NX-C18 110A column, while the remaining peptides were purified using a YMC-Pack C4 column. HPLC conditions typically include: Solvent A: 0.1% trifluoroacetic acid in 100% acetonitrile; Solvent B: 0.1% trifluoroacetic acid in 100% water; Gradient: 0.01 min: 20% (A), 80% (B); 25 min: 80% (A), 20% (B); 25.01 min: 100% (A), 0% (B); 30 min: stop; Flow rate: 1.0 ml / min; Wavelength: 220 nm; Volume: 20 μl. Molecular weights were confirmed by mass spectrometry using an Agilent-6125B liquid chromatography-mass spectrometer, and the peptide powders were lyophilized. The HPLC and MS spectra of each peptide are shown in the table below.

[0118] Table 3: Peptide sequences

[0119] Example 3: Evaluation of the degradation activity of polypeptide conjugates against Jun

[0120] 1. Evaluation of the Jun protein degradation effect of the peptide conjugate: The peptide conjugate was co-incubated with neonatal rat cardiomyocytes, and a concentration gradient or time gradient was set. Samples were collected and the degradation effect of the candidate peptide conjugate in neonatal rat cardiomyocytes was analyzed by immunoblotting (WB) and immunofluorescence (IF) assays, respectively.

[0121] The results for peptide conjugates J1-J7 are shown in Figure 18 . The WB results are shown in Figures 18A-18C , and the IF results are shown in Figures 18F-18G . Peptide J7 can gradually degrade Jun protein over time and concentration, demonstrating significant Jun protein degradation activity. The WB results for peptide conjugates J8-J11 and H1-H6 are shown in Figure 19 .

[0122] 2. Effect of peptide conjugates on mRNA: Peptide conjugates were added to neonatal rat cardiomyocytes for incubation. After the cells were treated with different concentrations of peptide conjugates, RNA was extracted from the cells. Fluorescence quantitative PCR experiments were used to detect changes in the gene c-jun and the internal reference gene GAPDH to evaluate the effect of the stable peptide degrader on c-jun gene transcription.

[0123] The qPCR results are shown in Figures 18D-18E , which show that degradation of Jun protein by J7 polypeptide does not affect changes in its mRNA level.

[0124] Example 4: In vivo evaluation of the efficacy of polypeptide conjugates

[0125] An HFpEF animal model was induced using the HFD+L-NAME method described in Example 1. Starting from the fifth week of HFpEF induction, mice in the treatment group were treated with J7 polypeptide, while mice in the control group were treated with the drug-free solvent, PBS. Mice that received a normal diet and water throughout the induction process served as negative controls. Mice were treated at five weeks of age. The treatment group received 100 mg / kg of J7 polypeptide intraperitoneally every other day, while the control group received an equal volume of PBS. All other treatments were identical.

[0126] The results, as shown in Figure 20, show that after treatment with the J7 polypeptide, diastolic function in HFpEF mice significantly improved, returning to the normal level of negative control mice; however, in the model control group mice that were not treated with J7, diastolic function continued to deteriorate. This indicates that the J7 polypeptide can have preventive and therapeutic effects on HFpEF in the mouse HFpEF model.

[0127] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A polypeptide conjugate or a pharmaceutically acceptable salt thereof, comprising: (a) a degradation-inducing ligand, (b) a cyclic peptide, and (c) a Jun protein targeting peptide; wherein: The Jun protein targeting peptide is selected from SEQ ID NO: 24 or SEQ ID NO:

25.

2. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The cyclic peptide has a structure shown in the general formula I: Cycle [isoD-X1-X2-Dap] (I) Wherein, isoD is isoaspartic acid, Dap is 2,3-diaminopropionic acid, X1 and X2 are each independently selected from natural amino acids, and the isoD and Dap form a cyclization; Preferably, X1 is E or V; Preferably, X2 is I or L.

3. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to claim 2, wherein: The cyclic peptide is Cyclo(isoD-EI-Dap) or Cyclo(isoD-VL-Dap); Preferably, the cyclic peptide is Cyclo(isoD-EI-Dap), and the Jun protein targeting peptide is SEQ ID NO: 24; Preferably, the cyclic peptide is Cyclo(isoD-VL-Dap), and the Jun protein targeting peptide is SEQ ID NO:

25.

4. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The degradation-inducing ligand is selected from E3 ubiquitin ligase ligands; Preferably, the degradation-inducing ligand is SEQ ID NO:

26.

5. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The degradation-inducing ligand is selected from LC3 ligands; Preferably, the degradation-inducing ligand is SEQ ID NO:

27.

6. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: Said (a), (b) and (c) are optionally connected to each other via a linker; Preferably, the degradation-inducing ligand and the cyclic peptide are directly connected or connected through a linker, and the cyclic peptide and the Jun protein targeting peptide are directly connected; Preferably, the linker is selected from aminocaproic acid or 12-aminolauric acid; Preferably, (a) and (b) are directly connected; Preferably, (a) and (b) are connected via a linker; Preferably, (b) and (c) are directly connected.

7. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein: The sequence of the polypeptide conjugate is shown in any one of SEQ ID NOs: 16-23 and 31-32.

8. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein: The polypeptide conjugate further comprises a nuclear localization sequence; Preferably, the polypeptide conjugate further comprises a nuclear localization sequence at its C-terminus; Preferably, the nuclear localization sequence is SEQ ID NO:

28.

9. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: The sequence of the polypeptide conjugate is shown in any one of SEQ ID NOs: 1-9 and 11-15.

10. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein: The polypeptide conjugate comprises an N-terminal blocking group, such as formyl, acetyl, or benzoyl.

11. The polypeptide conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein: The polypeptide conjugate comprises a detectable label; Preferably, the polypeptide conjugate comprises a detectable label at its N-terminus; Preferably, the detectable label is biotin.

12. A pharmaceutical composition comprising the polypeptide conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

13. Use of the polypeptide conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 12, in the preparation of a medicament for preventing and / or treating Jun-related diseases; Preferably, the related disease is heart failure with preserved ejection fraction (HFpEF).

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