Pharmaceutical composition comprising HTRA activator as active ingredient for preventing or treating degenerative diseases, and method for screening HTRA activity modulator

Abstract

The compound represented by chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof, according to one aspect, can be used as a prophylactic or therapeutic agent for degenerative diseases such as age-related macular degeneration (AMD) in which lack of HTRA1 activity is a factor in worsening the disease, cerebral amyloid angiopathy, hereditary stroke (CADASIL), and Alzheimer's disease (AD), and can be used as an adjuvant for efficiently and accurately screening an HTRA activity modulator without a change in temperature or addition of a substrate.

Description

Pharmaceutical composition for preventing or treating degenerative diseases containing HTRA activator as an active ingredient and screening method for HTRA activity modulator

[0001] The present invention relates to a pharmaceutical composition for preventing or treating degenerative diseases, which comprises an HTRA (High temperature requirement A serine protease) activator as an active ingredient, and a method for screening a degenerative HTRA activity modulator.

[0002] High temperature requirement A (HTRA) serine proteases are involved in protein quality control and cellular stress responses in various organisms, including humans, fungi, plants, mammals, and bacteria. They are particularly known to be involved in cellular stress responses and protein quality control in mammalian cells and microorganisms.

[0003] HTRA consists of a protease domain and a PDZ domain, and these multidomain complexes are characterized by oligomerization depending on their subtype and functional state. HTRA functions not only as a protease but also as a chaperone, playing a crucial role in alleviating protein-folding stress and regulating stress-related cellular responses.

[0004] Because HTRA is expressed ubiquitously in a wide range of organisms, it has been implicated in the pathogenesis of numerous diseases. For example, HTRA1 has been reported to degrade amyloid precursor protein (APP), amyloid β aggregates, and tau fibrils associated with Alzheimer's disease, while decreased HTRA2 activity is known to be associated with Parkinson's disease. Furthermore, altered human HTRA activity has been linked to arthritis, cancer, cerebral small vessel disease, age-related macular degeneration, and neurodegenerative diseases (Journal of Visualized Experiments Volume 2016, Issue 113, Article number e54136).

[0005] The activity of the above HTRA is induced by two different mechanisms: first, by allosteric effector binding to the PDZ domain, which induces cooperative remodeling into the active conformation; and second, by pepetide substrate binding to the active site, which promotes substrate-induced oligomerization. Through the above HTRA activity mechanism, HTRA activators with good cell penetration can be used to treat degenerative diseases caused by reduced HTRA activity.

[0006] However, the process of activating and switching the functional state of HTRA has not been fully elucidated, and research on HTRA-specific small-molecule activity modulators is insufficient.

[0007] HTRA enzyme activity remains low in the absence of an activator. Therefore, enzyme activity assays have traditionally involved adding excess substrate or heating the enzyme to 45°C to promote its reaction with the substrate. However, enzyme activity is significantly affected by temperature changes, with each enzyme operating most effectively at a specific temperature and diminishing at higher or lower temperatures (Campbell Biology, 4th ed., Figure 6.13, p101).

[0008] Accordingly, there is a need to develop a pharmaceutical composition for preventing or treating degenerative diseases using an HTRA activator and a method for efficiently screening an HTRA activity modulator at room temperature.

[0009] One aspect is to provide a compound represented by chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0010] Another aspect is to provide a pharmaceutical composition for preventing or treating a degenerative disease, comprising a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] Another aspect is to provide a health functional food for preventing or improving degenerative diseases, comprising a compound of chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Another aspect is to provide a reagent composition for HTRA activation, comprising a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another aspect provides a method for screening for an HTRA activity modulator, comprising the steps of: treating a sample in vitro with a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof; treating a sample treated with the compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof with a candidate substance; determining a level of HTRA (High temperature requirement A) activity; and comparing the level of HTRA activity with the level of HTRA activity of a sample not treated with the candidate substance.

[0014] Another aspect provides the use of a composition comprising a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating a degenerative disease.

[0015] Another aspect provides a method for preventing or treating a degenerative disease, comprising administering to a subject in need thereof a composition comprising an effective amount of a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Another aspect provides the use of a composition comprising a compound of the following formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of a degenerative disease.

[0017] In this specification, when a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless specifically stated otherwise.

[0018] One aspect provides a compound represented by the following chemical formula 1, an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

[0022] In one specific example, R1 and R2 may independently be a compound that is hydrogen or C1-C4 alkyl, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0023] In one specific example, R1 and R2 may independently be a compound that is hydrogen or methyl, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0024] One aspect provides a pharmaceutical composition for preventing or treating a degenerative disease, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0025] [Chemical Formula 1]

[0026]

[0027] In the above chemical formula 1,

[0028] R1 and R2 are independently hydrogen, C1-C10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

[0029] The term "alkyl" as used herein means a hydrocarbon having primary, secondary, tertiary and / or quaternary carbon atoms, which may be straight-chain, branched or a combination thereof.

[0030] In one specific example, the alkyl group has 1 to 10 carbon atoms (i.e., C1-C 10 alkyl), may have 1 to 4 carbon atoms (i.e., C1-C4 alkyl), or a methyl group.

[0031] For example, suitable alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-Methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), and octyl (-(CH2)7CH3), but are not limited thereto.

[0032] The term "alkenyl" as used herein refers to a substituent derived from an unsaturated hydrocarbon having 2 to 10 carbon atoms and a straight or branched chain having at least one carbon-carbon double bond. Examples thereof include, but are not limited to, vinyl, allyl, isopropenyl, and 2-butenyl.

[0033] The term "alkynyl" as used herein refers to a substituent derived from an unsaturated hydrocarbon having 2 to 10 carbon atoms and a straight or branched chain having at least one carbon-carbon triple bond. Examples thereof include, but are not limited to, ethynyl and 2-propynyl.

[0034] The term "isomer" as used herein refers to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is structurally or sterically different. Such isomers include structural isomers such as tautomers, which are formed by changing the position of hydrogen atoms, and stereoisomers. Stereoisomers include R or S isomers (enantiomers) having an asymmetric carbon center, and geometric isomers (trans, cis).

[0035] In the present invention, isomers of the compound represented by the above chemical formula 1 and mixtures thereof are also included in the scope of the present invention.

[0036] The term "hydrate" as used herein refers to a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Specifically, the hydrate of the compound represented by the above chemical formula 1 of the present invention may contain a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. For example, the hydrate may contain 1 equivalent or more, preferably 1 to 5 equivalents of water.

[0037] In one specific example, the hydrate can be prepared by crystallizing the compound represented by the chemical formula 1 of the present invention, an isomer or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

[0038] The term "solvate" as used herein refers to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and / or suitable for human administration.

[0039] The term "pharmaceutically acceptable salt" as used herein refers to a salt that is pharmaceutically acceptable and retains the desired pharmacological activity of the parent compound. The salt is not particularly limited, as long as it is pharmaceutically acceptable. For example, the pharmaceutically acceptable salt may be an acid addition salt formed with a pharmaceutically acceptable free acid. Specifically, acid addition salts can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc.The types of the above pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, Phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, malates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, and the like are not limited thereto. Pharmaceutically acceptable salts according to the present invention can be prepared by conventional methods.

[0040] As used herein, the term "degenerative disease" refers to a disease in which the function and structural damage of a tissue or organ gradually worsens over time. Specifically, the degenerative disease refers to a degenerative disease that occurs depending on the level of activity of HTRA (High Temperature Requirement A).

[0041] In one specific example, the degenerative disease may be age-related macular degeneration (AMD). The age-related macular degeneration (AMD) may be selected from the group consisting of wet macular degeneration, dry macular degeneration, geographic atrophy, and diabetic retinopathy, but is not limited thereto.

[0042] In one specific example, the degenerative disease may be a neurodegenerative disease. The above neurodegenerative diseases may be selected from the group consisting of dementia, Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, cerebral amyloid angiopathy, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, hereditary stroke (CADASIL), amyotrophic lateral sclerosis, spinocerebellar atrophy, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia, but are not limited thereto.

[0043] The above HTRA is a type of serine protease involved in the composition of the extracellular matrix, cell proliferation, and aging. HTRA activity levels are associated with several diseases, including Duchenne muscular dystrophy (Bakay et al. 2002, Neuromuscul. Disord. 12: 125-141), arthritis such as osteoarthritis (Grau et al. 2006, JBC 281: 6124-6129), cancer, familial ischemic cerebral small-vessel disease, and age-related macular degeneration, as well as Parkinson's disease and Alzheimer's disease. For example, HTRA1 inhibits signaling by the TGF β family of growth factors (Oka et al., 2004, Development 131: 1041-1053). HTRA1 can cleave amyloid precursor protein (APP), and HTRA1 inhibitors cause accumulation of Aβ peptides in cultured cells. Therefore, decreased HTRA1 activity is associated with Alzheimer's disease (Grau et al., 2005, Proc. Nat. Acad. Sci. USA. 102:6021-6026).

[0044] The term "prevention" as used herein may include, without limitation, any action that can block symptoms caused by a degenerative disease, or inhibit or delay its progression, using the composition of the present invention. The term "treatment" as used herein refers to a series of activities performed to alleviate and improve the desired disease.

[0045] In one specific example, the pharmaceutical composition may activate HTRA (High temperature requirement A).

[0046] As used herein, the term “HTRA (High temperature requirement A)” refers to one of the serine proteases that plays a role in protein folding problems and degradation of damaged proteins. HTRA is involved in protein quality control and cellular stress responses in various organisms, including humans, fungi, plants, mammals, and bacteria.

[0047] In the case of microorganisms, HTRA (High temperature requirement A) is known to be found in highly pathogenic and antibiotic-resistant species such as Helicobacter pylori species or Chlamydia species, and is known to be involved in the penetration of host cells and the survival of microorganisms in heat-stress or acid-stress environments.

[0048] In humans, HTRA exists in four isoforms: HTRA1, HTRA2, HTRA3, and HTRA4. HTRA1 is known to be localized in the cell membrane, cytoplasm, and nucleus, while HTRA2 is known to be located within mitochondria. The human HTRA (high temperature requirement A) family is a ubiquitous PDZ-protease family involved in maintaining protein homeostasis in the extracellular space by combining the dual functions of protease and chaperone.

[0049] In one specific example, the HTRA may be one or more selected from HTRA1 and HTRA2.

[0050] The pharmaceutical composition according to the present invention may further include an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0051] Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0052] The pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0053] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included.

[0054] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0055] Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0056] Additionally, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., and may be appropriately selected by a skilled medical professional. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0057] The above pharmaceutical composition can be administered to mammals such as rats, mice, livestock, and humans via various routes. For example, it can be administered orally, rectally, intravenously, intramuscularly, subcutaneously, intratracheally, intrauterinely, or intracerebroventricularly, but is not limited thereto.

[0058]

[0059] Another aspect provides a health functional food for preventing or improving degenerative diseases, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0060] [Chemical Formula 1]

[0061]

[0062] In the above chemical formula 1,

[0063] R1 and R2 are independently hydrogen or C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl.

[0064] The term "improvement" as used herein means alleviating or alleviating the symptoms of a degenerative disease by administering, ingesting, or applying the pharmaceutical composition or food composition of the present invention to a subject suffering from a degenerative disease.

[0065] The term "health functional food" as used herein refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body as defined in the Health Functional Food Act, and the term "functionality" as used herein refers to ingestion for the purpose of obtaining a beneficial effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. Specifically, it refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functionality useful to the human body. There are no particular limitations on the types of the above foods, and all health functional foods in the conventional sense are included.

[0066] The health functional food of the present invention can be manufactured using methods commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. Furthermore, the health functional food can be manufactured in any formulation recognized as a health functional food without limitation. Specifically, the health functional food can be provided in the form of powder, granules, tablets, capsules, syrup, or beverage.

[0067] The above health functional food is used in combination with other foods or food additives in addition to the compound represented by the above chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof, which is an effective ingredient, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredient can be appropriately determined depending on the purpose of use, for example, prevention, health, or therapeutic treatment.

[0068] The matters mentioned in the pharmaceutical composition and health functional food of the present invention apply equally unless they are contradictory.

[0069] Another aspect provides a method for preventing or treating a degenerative disease, comprising administering to a subject in need thereof a compound of the above-described chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0070] Another aspect provides the use of a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for use in the prevention or treatment of a degenerative disease.

[0071] In the above method or use, the detailed description of the pharmaceutical composition described above may be applied.

[0072]

[0073] Another aspect provides a reagent composition for HTRA activation, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof as an active ingredient.

[0074] [Chemical Formula 1]

[0075]

[0076] In the above chemical formula 1,

[0077] R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

[0078] In one specific example, the reagent composition may contain a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof at a concentration of 10 μM to 100 μM. For example, the reagent composition comprises a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof at a concentration of 10 μM to 20 μM, 10 μM to 30 μM, 10 μM to 40 μM, 10 μM to 50 μM, 10 μM to 60 μM, 10 μM to 70 μM, 10 μM to 80 μM, 10 μM to 90 μM, 10 μM to 100 μM, 20 μM to 30 μM, 20 μM to 40 μM, 20 μM to 50 μM, 20 μM to 60 μM, 20 μM to 70 μM, 20 μM to 80 μM, 20 μM to 90 μM, 20μM to 100μM, 30μM to 40μM, 30μM to 50μM, 30μM to 60μM, 30μM to 70μM, 30μM to 80μM, 30μM to 90μM, 30μM to 100μM, 40μM to 50μM, 40μM to 60μM, 40μM to 70μM, 40μM to 80μM, 40μM to 90μM, 40μM to 100μM, 50μM to 60μM, 50μM to 70μM, 50μM to 80μM, 50μM to 90μM, 50μM to 100μM, 60μM to 70μM, It may be included at a concentration of, but is not limited to, 60 μM to 80 μM, 60 μM to 90 μM, 60 μM to 100 μM, 70 μM to 80 μM, 70 μM to 90 μM, 70 μM to 100 μM, 80 μM to 90 μM, 80 μM to 100 μM, 90 μM to 100 μM.

[0079] The above enzyme activity assay refers to measuring the activity of a specific enzyme by measuring the substrate reduction rate or product increase rate.

[0080] The HTRA activating reagent composition comprising the compound of the above chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or an acceptable salt thereof binds to an allosteric site of the HTRA enzyme, thereby modifying the conformation of the enzyme, thereby enabling an HTRA enzyme activity assay without a change in temperature or addition of a substrate.

[0081]

[0082] Another aspect provides a method for screening for an HTRA activity modulator, comprising: a step of activating HTRA by treating a sample containing HTRA (High temperature requirement A) in vitro with a compound of the following formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof; a step of treating a sample containing the activated HTRA with a candidate substance; and a step of comparing the HTRA activity level of the sample treated with the candidate substance with the HTRA activity level of an untreated sample.

[0083] [Chemical Formula 1]

[0084]

[0085] In the above chemical formula 1,

[0086] R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

[0087] In one specific example, the comparing step may include a step of selecting a candidate substance that changes the level of HTRA activity as an HTRA modulator.

[0088] In one specific example, the screening method may be characterized by selecting a sample as an HTRA activator when the HTRA activity level of a sample treated with the candidate substance is increased compared to the HTRA activity level of a sample not treated with the candidate substance.

[0089] In one specific example, the screening method may be characterized by selecting as an HTRA inhibitor when the HTRA activity level of a sample treated with the candidate substance is reduced compared to the HTRA activity level of a sample not treated with the candidate substance.

[0090] The step of confirming the above HTRA activity level may use a peptide probe for HTRA detection based on HTRA activity, but is not limited thereto. Specifically, the peptide probe for HTRA detection based on HTRA activity means that it is characterized by detecting human HTRA1, human HTRA2, and microbial-derived HTRA based on activity.

[0091] In one specific example, the sample may be selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

[0092] Another aspect provides the use of a composition comprising a compound of the following formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating a degenerative disease.

[0093] [Chemical Formula 1]

[0094]

[0095] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

[0096] Another aspect provides a method for preventing or treating a degenerative disease, comprising administering to a subject in need thereof a composition comprising an effective amount of a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. In formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

[0097] Another aspect provides the use of a composition comprising a compound of formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of a degenerative disease. In formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

[0098] The terms and methods described for the above inventions apply equally to each invention.

[0099] The compound represented by chemical formula 1 according to one aspect, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof can be used as an agent for preventing or treating degenerative diseases such as macular degeneration (AMD), cerebral amyloid angiopathy, hereditary stroke (CADASIL), and Alzheimer's disease (AD), in which lack of HTRA1 activity is a factor in the aggravation of the disease, and can be used as an auxiliary agent for efficiently and accurately screening for HTRA activity modulators without temperature change or substrate addition.

[0100] Figure 1 is a graph showing the results of the HTRA1 activity assay of compounds E01 to E12 obtained through Atomwise.

[0101] Figure 2 is a graph showing the activity change of HTRA1 according to the E09 dose: Figure 2(a) is a graph showing the activity change of HTRA1 when the E09 dose is 1 nM to 1 μM, Figure 2(b) is a graph showing the activity change of HTRA1 when the E09 dose is 1 nM to 10 μM, Figure 2(c) is a graph showing the activity change of HTRA1 when the E09 dose is 0 μM to 100 μM, and Figure 2(d) is a graph showing the activity of HTRA1 when E09 is injected (final concentration 10 μM) and when it is not injected.

[0102] Figure 3 is a graph showing the activity change of HTRA2 according to the E09 dose: Figure 3(a) is a graph showing the activity change of HTRA2 when the E09 dose is 1 nM to 1 μM, and Figure 3(b) is a graph showing the activity change of HTRA2 when the E09 dose is 1 nM to 10 μM.

[0103] Figure 4 is a photograph of HTRA1 activity observed under a fluorescence microscope after treating HeLa cells with HTRA1 probe (DS-1-94, final concentration 5 μM) and E09 (HTRA activator, final concentration 10 μM).

[0104] Figure 5 is a graph measuring the enzyme activity of HTRA1 using caseine, known as a substrate of HTRA enzyme, or PDZ-opt (GQYYFV) and E09, known as peptides that activate HTRA enzyme.

[0105] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0106]

[0107] Example 1. Discovery of HTRA activator

[0108] We conducted an activity screening of 72 compounds acquired through Atomwise, an AI-based compound design company. These 72 compounds were designed using the active site structure of DegP (Degradation of periplasmic proteins), a bacterial homolog of the HTRA family.

[0109] HTRA activity was confirmed through high-throughput screening using H2-Opt, a commercially available fluorogenic substrate.

[0110] Specifically, using a 96-well plate, purified recombinant HTRA1 protein (50 ng) and compounds (final concentration 10 μM) were added to 80 μL of assay buffer per well and left at room temperature for 30 minutes. 20 μL of H2-OPT solution (final concentration 6.25 μM) was added and mixed, and the resulting fluorescence signal was measured using an M5 microplate reader from molecular devices. When measuring the fluorescence signal, the kinetic mode was set to excitation 328 nm, emission 393 nm, and fluorescence was measured at 37 °C for 3 hours at 30-second intervals. oFluorescence signal changes were measured in C. The results of HTRA activity measurement through high-throughput screening are shown in Fig. 1.

[0111] Figure 1 is a graph showing the results of the HTRA1 activity assay of compounds E01 to E12 obtained through Atomwise.

[0112] As shown in Fig. 1, among compounds E01 to E12, E09 was found to significantly activate HTRA1. It was confirmed that E09 exhibited excellent activation ability not only for HTRA1 but also for HTRA2.

[0113] For reference, the structure of the E09 compound is as shown in Chemical Formula 1 below.

[0114] [Chemical Formula 1]

[0115]

[0116] The E09 compound was purchased from Enamine (NJ, USA) (catalog number Z1571762517) and used.

[0117]

[0118] Example 2. Measurement of E09 dose-dependent activity of HTRA1 and HTRA2

[0119] The activity change according to the dose of E09 for HTRA1 and HTRA2 was confirmed.

[0120] Specifically, different concentrations of E09 (0.01, 0.1, 1, 10, 100 μM) were added to purified recombinant HTRA1 and HTRA2, and the activities of HTRA1 and HTRA2 were measured in the same manner as the high throughput screening of Example 1. The fluorescence signal at the end of the measurement (3 hours) was measured for each concentration. The graphs measuring the fluorescence signals of HTRA1 and HTRA2 are shown in Figs. 2 and 3.

[0121] Figure 2 is a graph showing the activity change of HTRA1 according to the E09 dose: Figure 2(a) is a graph showing the activity change of HTRA1 when the E09 dose is 1 nM to 1 μM, Figure 2(b) is a graph showing the activity change of HTRA1 when the E09 dose is 1 nM to 10 μM, Figure 2(c) is a graph showing the activity change of HTRA1 when the E09 dose is 0 μM to 100 μM, and Figure 2(d) is a graph showing the activity of HTRA1 when E09 was added (final concentration 10 μM) and when it was not added.

[0122] Figure 3 is a graph showing the activity change of HTRA2 according to the E09 dose: Figure 3(a) is a graph showing the activity change of HTRA2 when the E09 dose is 1 nM to 1 μM, and Figure 3(b) is a graph showing the activity change of HTRA2 when the E09 dose is 1 nM to 10 μM.

[0123] As shown in Figures 2 and 3, it was confirmed that as the E09 dose increased, the activities of HTRA1 and HTRA2 increased. This means that E09 activates HTRA1 and HTRA2 in a concentration-dependent manner and also activates HTRA1 in human blood samples.

[0124]

[0125] Example 3. Confirmation of HTRA1 activity in the presence or absence of E09 in HeLa cells.

[0126] To confirm whether HTRA1 activity increases when cells are treated with E09, human adenocarcinoma HeLa cells were treated with an HTRA1 activity-based probe (DS-1-94, final concentration 5 μM) and E09 (HTRA activator, final concentration 10 μM) to confirm the activity of HTRA 1 in the presence or absence of E09.

[0127] Specifically, cells treated with HTRA1 activity-based probe (DS-1-94) and E09 (HTRA activator) were placed in a 37°C incubator for 1 hour. Thereafter, cells were observed under a fluorescence microscope to determine how the probe reacted in living cells, and the results are shown in Figure 4.

[0128] Figure 4 is a photograph of HTRA1 activity observed under a fluorescence microscope after treating HeLa cells with an HTRA1 activity-based probe (DS-1-94, final concentration 5 μM) and E09 (HTRA activator, final concentration 10 μM).

[0129] As shown in Fig. 4, it was confirmed that the fluorescence signal was clearly increased in cells treated with the activator (E09). This means that when E09 is treated to cells, the activity of HTRA1 increases.

[0130]

[0131] Example 4. Measurement of changes in HTRA activity according to the addition of HTRA activator

[0132] 4.1 Preparation of HTRA activity-based probe

[0133] An HTRA activity-based probe was manufactured using the method known in the inventor's prior registered patent KR 10-2302971.

[0134] Specifically, the hydrophobic functional groups of Val and Leu were selected as amino acid functional groups located at P1 of the substrate-binding domain (or active domain) of HTRA, which are well conserved among HTRA proteins, to construct the P1-V tetrapeptide probe series (IRRV-probe, FIMV-probe, FKLV-probe, FKGV-probe, ASPV-probe, LFLV-probe, MTP-LFLV-probe, and TPP-LFLV-probe) and the P1-L tetrapeptide probe series (FIML-probe, FKLL-probe, FKGL-probe, ASPL-probe, and LFLL-probe). In addition, in order to confirm the subfamily selectivity, the P2, P3, and P4 positions of the P1-V tetrapeptide probe series and the P1-L tetrapeptide probe series were designed by changing various types of amino acid functional groups. Thirteen types of tetrapeptide probes were synthesized based on the HTRA substrate amino acid sequences reported through previous research results.

[0135] 4.2 Comparison of HTRA enzyme activity by HTRA activator

[0136] HTRA activity was measured using the HTRA activity-based probe synthesized by the method of Example 4.1 and E09. The change in HTRA1 activity was measured when caseine, known as a substrate of HTRA enzyme, or PDZ-opt (GQYYFV), known as a peptide that activates HTRA enzyme, was added, and when E09 was added.

[0137] Specifically, 2 μg of ß-caseine or PDZ-opt (GQYYFV) or 10 μM of E09 was added to the HTRA1 enzyme according to the method of Example 1, and the mixture was left at room temperature for 15 minutes. Then, HTRA1 activity was measured at 37°C for 3 hours according to the same method as Example 1, and the results are shown in Fig. 5.

[0138] Figure 5 is a graph measuring the enzyme activity of HTRA1 using caseine, known as a substrate of HTRA enzyme, or PDZ-opt(GQYYFV) and E09, known as peptides that activate HTRA enzyme.

[0139] As shown in Fig. 5, it was confirmed that E09 activates HTRA1 enzyme significantly more than caseine or PDZ-opt(GQYYFV) at room temperature.

[0140] These results imply that E09 can effectively measure HTRA activity without temperature change or substrate addition, even with low doses of HTRA enzyme.

Claims

1. A pharmaceutical composition for preventing or treating a degenerative disease, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

2. In claim 1, A pharmaceutical composition wherein the above R1 and R2 are independently hydrogen or a C1-C4 alkyl compound, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

3. In claim 1, A pharmaceutical composition wherein the above R1 and R2 are independently hydrogen or methyl, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition according to claim 1, wherein the degenerative disease is age-related macular degeneration (AMD).

5. A pharmaceutical composition according to claim 4, wherein the macular degeneration is selected from the group consisting of wet macular degeneration, dry macular degeneration, geographic atrophy, and diabetic retinopathy.

6. In claim 1, the degenerative disease is selected from the group consisting of dementia, Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, cerebral amyloid angiopathy, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, CADASIL, amyotrophic lateral sclerosis, spinocerebellar atrophy, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia. Pharmaceutical composition.

7. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition activates HTRA (High temperature requirement A).

8. A pharmaceutical composition according to claim 7, wherein the HTRA is at least one selected from HTRA1 and HTRA2.

9. A health functional food for preventing or improving degenerative diseases, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

10. A reagent composition for HTRA activation, comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

11. A reagent composition for HTRA activation according to claim 10, wherein the reagent composition comprises a compound of chemical formula 1 at a concentration of 10 μM to 100 μM.

12. A step of activating HTRA by treating a sample containing HTRA (High temperature requirement A) in vitro with a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof; A step of treating a candidate substance in a sample containing the activated HTRA; and A method for screening for an HTRA activity modulator, comprising: a step of comparing the HTRA activity level of a sample treated with the candidate substance with the HTRA activity level of an untreated sample; [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It's alkynyl.

13. A method for screening an HTRA activity modulator according to claim 12, wherein the comparing step includes a step of selecting a candidate substance that changes the HTRA activity level as an HTRA modulator.

14. A method for screening an HTRA activity modulator according to claim 12, wherein the sample is selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

15. Use of a composition comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating a degenerative disease: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

16. A method for preventing or treating a degenerative disease, comprising administering to a subject in need thereof a composition comprising an effective amount of a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

17. Use of a composition comprising a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient for use in the manufacture of a medicine for the prevention or treatment of degenerative diseases: [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 It is alkynyl.

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