Pharmaceutical composition for the prevention or treatment of degenerative diseases containing HTRA activator as an active ingredient and Screening Method for HTRA activity modulators

Abstract

Compounds represented by Chemical Formula 1 according to one aspect, stereoisomers thereof, solvates thereof, hydrates thereof, or pharmaceutically acceptable salts thereof can be used as preventive or therapeutic agents for degenerative diseases such as macular degeneration (AMD), cerebral amyloid angiopathy, hereditary stroke (CADASIL), and Alzheimer's disease (AD), in which a lack of HTRA1 activity is a factor in the exacerbation of the disease, and can be used as adjuvants for efficiently and accurately screening HTRA activity modifiers without temperature changes or substrate additions.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative diseases comprising an HTRA (High temperature requirement A serine protease) activator as an active ingredient, and a method for screening degenerative HTRA activity modifiers. Background Technology

[0002] HTRA (High temperature requirement A) serine proteases are involved in protein quality control and cellular stress responses in various organisms, including humans, fungi, plants, mammals, and bacteria. In particular, they are 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 this multi-binding domain complex is characterized by oligomerization depending on their subtype and functional states. By functioning as both a protease and a chaperone, HTRA plays an important role in alleviating protein-folding stress and regulating stress-related cellular responses.

[0004] Because HTRA is expressed throughout a wide range of organisms, it is associated with the pathogenesis of many diseases. For example, it has been reported that HTRA1 degrades amyloid precursor protein (APP), amyloid β-aggregates, and tau fibrils associated with Alzheimer's disease, and reduced HTRA2 activity is known to be associated with Parkinson's disease. Furthermore, changes in human HTRA activity are associated with arthritis, cancer, cerebrovascular 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 the binding of an allosteric effector to the PDZ domain, which induces cooperative remodeling through active conformation; and second, by the binding of a pepetide substrate to the active site, which promotes substrate-induced oligomerization. Through the above HTRA activation mechanism, an HTRA activator with good cell permeability can be used to treat degenerative diseases caused by reduced HTRA activity.

[0006] However, the processes of activating and switching the functional states of HTRA have not been fully elucidated, and research on HTRA-specific small molecule activity regulators is lacking.

[0007] Since the activity of HTRA enzymes remains at a low level in the absence of an activator, conventional methods to perform enzyme activity assays involved adding an excess amount of substrate or promoting the reaction with the substrate at 45°C through heating. However, enzyme activity is significantly affected by temperature changes; each enzyme acts most effectively at a specific temperature, and its activity decreases at higher or lower temperatures (Campbell Biology 4th ed. Figure 6.13, p101).

[0008] Accordingly, there is a need to develop pharmaceutical compositions for the prevention or treatment of degenerative diseases using HTRA activators, as well as a method for efficiently screening HTRA activity modifiers at room temperature. The problem to be solved

[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 provides a pharmaceutical composition for the prevention or treatment of degenerative diseases, comprising as an active ingredient a compound of Formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

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

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

[0013] Another aspect provides a method for screening HTRA activity modifiers, 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 said compound of Formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof with a candidate substance; determining the level of HTRA (High temperature requirement A) activity; and comparing said HTRA activity level with the level of HTRA activity of a sample not treated with the candidate substance. means of solving the problem

[0014] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0015] One aspect provides a compound represented by Chemical Formula 1, its isomer, its solvate, its hydrate, or a pharmaceutically acceptable salt thereof.

[0016] [Chemical Formula 1]

[0017]

[0018] 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 alkinil.

[0019] In one embodiment, R1 and R2 may independently be hydrogen or a C1-C4 alkyl compound, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0020] In one embodiment, 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.

[0021] One aspect provides a pharmaceutical composition for the prevention or treatment of degenerative diseases, comprising as an active ingredient a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0022] [Chemical Formula 1]

[0023]

[0024] In the above chemical formula 1,

[0025] R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It is alkinil.

[0026] The term "alkyl" in this specification is a hydrocarbon having primary, secondary, tertiary and / or quaternary carbon atoms, and may be straight-chain, branched, or a combination thereof.

[0027] In one embodiment, the alkyl group comprises 1 to 10 carbon atoms (i.e., C1-C 10 It may have alkyl), 1 to 4 carbon atoms (i.e., C1-C4 alkyl), or a methyl group.

[0028] 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), and 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 It may be (-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 is not limited thereto.

[0029] The term "alkenyl" in this specification is a substituent derived from a straight-chain or side-chain unsaturated hydrocarbon having 2 to 10 carbon atoms and having one or more carbon-carbon double bonds. Examples may include, but are not limited to, vinyl, allyl, isopropenyl, 2-butenyl, etc.

[0030] The term “alkynyl” in this specification is a substituent derived from a straight-chain or side-chain unsaturated hydrocarbon having 2 to 10 carbon atoms and having one or more carbon-carbon triple bonds. For example, it may be ethynyl or 2-propynyl, but is not limited thereto.

[0031] The term "isomer" in this specification 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 stereochemically different. These isomers include structural isomers, such as tautomers, which are formed by a change in the position of hydrogen atoms, and stereoisomers. Stereoisomers include R or S isomers having an asymmetric carbon center (optical isomers, enantiomers), geometric isomers (trans, cis), etc.

[0032] In the present invention, isomers of the compound represented by the above chemical formula 1 and mixtures thereof are also included within the scope of the present invention.

[0033] The term "hydrate" in this specification 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 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 at least 1 equivalent, preferably 1 to 5 equivalents of water.

[0034] In one embodiment, the hydrate may be prepared by crystallizing a compound represented by Formula 1 of the present invention, an isomer, or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

[0035] The term "solvate" in this specification refers to a compound of the present invention or a salt thereof comprising stoichiometric or non-stoichiometric amounts of solvent bound by non-covalent intermolecular forces. Preferred solvents thereof include volatile, non-toxic, and / or solvents suitable for administration to humans.

[0036] The term “pharmaceuticalally acceptable salt” in this specification means a salt that is pharmaceutically acceptable and retains the desirable pharmacological activity of the parent compound. The salt is not particularly limited as long as it is pharmaceutically acceptable. For example, an acid addition salt formed by a pharmaceutically acceptable free acid may be used as the pharmaceutically acceptable salt. Specifically, acid addition salts can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromide, hydroiodide, nitrous acid, phosphoric acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkandioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., non-toxic 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-mentioned pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chloride, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, malieates, butyne-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, and phthalates. The present invention includes, but is not limited to, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc. Pharmaceutically acceptable salts according to the present invention can be prepared by conventional methods.

[0037] In this specification, the term "degenerative disease" refers to a disease in which functional and structural damage to a tissue or organ gradually deteriorates 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).

[0038] In one embodiment, 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.

[0039] In one embodiment, the degenerative disease may be a neurodegenerative disease. The above-mentioned 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.

[0040] The aforementioned HTRA is a type of serine protease involved in the composition of the extracellular matrix, cell proliferation, and aging. Levels of HTRA activity are associated with 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 cerebrovascular disease, and age-related macular degeneration, as well as various diseases including Parkinson's disease and Alzheimer's disease. For example, HTRA1 is related to the growth factor TGF It inhibits signal transduction by the family (Reference [Oka et al., 2004, Development 131:1041-1053]). HTRA1 can cleave amyloid precursor protein (APP), and HTRA1 inhibitors cause the accumulation of Aβ peptides in cultured cells. Consequently, reduced HTRA1 activity is associated with Alzheimer's disease (Reference [Grau et al., 2005, Proc. Nat. Acad. Sci. USA. 102:6021-6026]).

[0041] The term "prevention" in this specification may be included without limitation as long as it is any act capable of blocking symptoms caused by a degenerative disease, or inhibiting or delaying its progression, using the composition of the present invention. The term "treatment" in this specification refers to a series of activities performed for the alleviation and improvement of a target disease.

[0042] In one embodiment, the pharmaceutical composition may activate HTRA (High temperature requirement A).

[0043] The term "HTRA (High temperature requirement A)" in this specification refers to one of the serine proteases that plays a role in protein folding problems and the 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.

[0044] In the case of microorganisms, HTRA (High temperature requirement A) is known to be found in species with high pathogenicity and high antibiotic resistance, such as Helicobacter pylori or Chlamydia species, and is known to be involved in invasive action against host cells and in the survival of microorganisms in heat stress or acid stress environments.

[0045] In humans, HTRA exists in four isoforms: HTRA1, HTRA2, HTRA3, and HTRA4. HTRA1 is known to be located in the cell membrane, cytoplasm, and nucleus, while HTRA2 is known to be located inside the mitochondria. The human HTRA (high temperature requirement A) family is a universally expressed PDZ-protease involved in maintaining protein homeostasis in the extracellular region by combining the dual functions of a protease and a chaperone.

[0046] In one embodiment, the HTRA may be one or more selected from HTRA1 and HTRA2.

[0047] The pharmaceutical composition according to the present invention may further include a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions.

[0048] 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0049] The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, each according to conventional methods. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0050] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing the compound with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, for example, humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0051] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0052] Witepsol, Macrogol, Tween 61, Cacao, Laurin, Glycerozelatin, etc. can be used as bases for suppositories.

[0053] Furthermore, 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. Accordingly, the above dosage does not limit the scope of the present invention in any way.

[0054] The above pharmaceutical composition may be administered to mammals, such as rats, mice, livestock, and humans, by various routes. For example, it may be administered orally, rectally or intravenously, intramuscularly, subcutaneously, by bronchial inhalation, intrauterine dura mater, or intracerebroventricular injection, but is not limited thereto.

[0055] Another aspect provides a health functional food for the prevention or improvement of degenerative diseases, comprising as an active ingredient a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0056] [Chemical Formula 1]

[0057]

[0058] In the above chemical formula 1,

[0059] R1 and R2 are independently hydrogen or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkenyl.

[0060] The term "improvement" in this specification includes the reduction or alleviation of symptoms of a degenerative disease by administering, ingesting, or applying the pharmaceutical composition or food composition of the present invention to an individual suffering from a degenerative disease.

[0061] The term "health functional food" in this specification refers to a food manufactured and processed using raw materials or ingredients having functional properties beneficial to the human body in accordance with the Act on Health Functional Foods. The term "functionality" in this specification refers to consumption for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. Specifically, it refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functional properties beneficial to the human body. There are no special restrictions on the types of the above foods, and they include all health functional foods in the conventional sense.

[0062] The health functional food of the present invention can be manufactured by methods commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during such manufacturing. Furthermore, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. Specifically, the health functional food may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage.

[0063] The above-mentioned health functional food may be used in combination with other foods or food additives in addition to the active ingredient, the compound represented by Chemical Formula 1, its stereoisomer, its solvate, its hydrate, or its pharmaceutically acceptable salt, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its intended use, for example, for prevention, health, or therapeutic treatment.

[0064] The matters mentioned in the pharmaceutical composition and health functional food of the present invention shall apply equally unless they contradict each other.

[0065] Another aspect provides a method for preventing or treating a degenerative disease comprising the step of administering the compound of Formula 1, its stereoisomer, its solvate, its hydrate, or its pharmaceutically acceptable salt to a subject in need.

[0066] Another aspect provides a use of the compound of Formula 1, its stereoisomers, its solvates, its hydrates, or its pharmaceutically acceptable salts for use in the manufacture of a medicament for the prevention or treatment of degenerative diseases.

[0067] In the above method or use, the detailed description of the pharmaceutical composition described above may be applied.

[0069] Another aspect provides a reagent composition for HTRA activation comprising, as an active ingredient, a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof.

[0070] [Chemical Formula 1]

[0071]

[0072] In the above chemical formula 1,

[0073] R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It is alkinil.

[0074] In one embodiment, 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 above reagent composition comprises a compound of Formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof in an amount 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 concentrations of 60μM to 80μM, 60μM to 90μM, 60μM to 100μM, 70μM to 80μM, 70μM to 90μM, 70μM to 100μM, 80μM to 90μM, 80μM to 100μM, and 90μM to 100μM, but is not limited thereto.

[0075] The aforementioned enzyme activity assay refers to measuring the degree of enzyme activity through methods such as measuring the rate of substrate reduction or the rate of product increase of a specific enzyme.

[0076] A reagent composition for HTRA activation comprising the compound of Formula 1, its stereoisomer, its solvate, its hydrate, or an acceptable salt thereof enables an HTRA enzyme activity assay without temperature changes or substrate addition by modifying the conformation of the enzyme through binding to the allosteric site of the HTRA enzyme.

[0078] Another aspect provides a method for screening HTRA activity modifiers, comprising the steps of: activating HTRA in vitro by treating a sample containing HTRA (High temperature requirement A) with a compound of Formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof; treating the sample containing the activated HTRA with a candidate substance; and comparing the level of HTRA activity of the sample treated with the candidate substance with the level of HTRA activity of an untreated sample.

[0079] [Chemical Formula 1]

[0080]

[0081] In the above chemical formula 1,

[0082] R1 and R2 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It is alkinil.

[0083] In one embodiment, the comparing step may include the step of selecting a candidate substance that changes the HTRA activity level as an HTRA modulator.

[0084] In one embodiment, the screening method may be characterized by selecting an HTRA activator when the HTRA activity level of a sample treated with a candidate substance is higher than the HTRA activity level of a sample not treated with a candidate substance.

[0085] In one embodiment, the screening method may be characterized by selecting an HTRA inhibitor when the HTRA activity level of a sample treated with the candidate substance is lower than the HTRA activity level of a sample not treated with the candidate substance.

[0086] The step of confirming the level of HTRA activity above 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.

[0087] In one embodiment, the sample may be selected from the group consisting of cells, tissues, blood, serum, urine, and saliva. Effects of the invention

[0088] Compounds represented by Chemical Formula 1 according to one aspect, stereoisomers thereof, solvates thereof, hydrates thereof, or pharmaceutically acceptable salts thereof can be used as preventive or therapeutic agents for degenerative diseases such as macular degeneration (AMD), cerebral amyloid angiopathy, hereditary stroke (CADASIL), and Alzheimer's disease (AD), in which a lack of HTRA1 activity is a factor in the exacerbation of the disease, and can be used as adjuvants for efficiently and accurately screening HTRA activity modifiers without temperature changes or substrate additions. Brief explanation of the drawing

[0089] Figure 1 is a graph showing the results of the HTRA1 activity assay of compounds E01 to E12 obtained through Atomwise. Figure 2 is a graph showing the change in activity of HTRA1 according to the E09 dose: Figure 2(a) is a graph showing the change in activity of HTRA1 when the E09 dose is 1 nM to 1 μM, Figure 2(b) is a graph showing the change in activity of HTRA1 when the E09 dose is 1 nM to 10 μM, Figure 2(c) is a graph showing the change in activity 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 added (final concentration 10 μM) and when it is not added. Figure 3 is a graph showing the change in activity of HTRA2 according to the E09 dose: Figure 3(a) is a graph showing the change in activity of HTRA2 when the E09 dose is 1 nM to 1 μM, and Figure 3(b) is a graph showing the change in activity of HTRA2 when the E09 dose is 1 nM to 10 μM. Figure 4 is a fluorescence microscope image showing the activity of HTRA1 after treating HeLa cells with HTRA1 probe (DS-1-94, final concentration 5 μM) and E09 (HTRA activator, final concentration 10 μM). Figure 5 is a graph showing the enzyme activity of HTRA1 using caseine, known as a substrate of the HTRA enzyme, or PDZ-opt (GQYYFV) and E09, known as peptides that activate the HTRA enzyme. Specific details for implementing the invention

[0090] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0091] Example 1. Discovery of HTRA activators

[0092] Activity screening was performed on 72 compounds obtained through Atomwise, an AI-based compound design company. The 72 compounds were designed using the active site structure of DegP (Degradation of periplasmic proteins), a bacterial homolog of the HTRA family.

[0093] HTRA activity was confirmed through high-throughput screening using H2-Opt, a commercially available fluorogenic substrate.

[0094] Specifically, using a 96-well plate, purified recombinant HTRA1 protein (50 ng) and the compound (final concentration 10 μM) were added to 80 μL of assay buffer per well and incubated at room temperature for 30 minutes. Immediately after adding and mixing 20 μL of H2-OPT solution (final concentration 6.25 μM), the generated fluorescence signal was measured using a Molecular Devices M5 microplate reader. For fluorescence measurement, the kinetic mode was set to excitation 328 nm and emission 393 nm, and measurements were taken at 30-second intervals for 3 hours. o Changes in fluorescence signal were measured in C. The results of HTRA activity measurement through high-throughput screening are shown in Figure 1.

[0095] Figure 1 is a graph showing the results of the HTRA1 activity assay of compounds E01 to E12 obtained through Atomwise.

[0096] As shown in Figure 1, among compounds E01 to E12, it was discovered that E09 clearly activates HTRA1. It was confirmed that E09 exhibits excellent activation ability in both HTRA1 and HTRA2.

[0097] delete

[0098] Compound E09 was purchased from Enamine (NJ, USA) (Catalog number Z1571762517) and used.

[0099] delete

[0100] delete

[0102] Example 2. Measurement of E09 dose-dependent activity of HTRA1 and HTRA2

[0103] The dose-dependent changes in activity of HTRA1 and HTRA2 were examined for each drug.

[0104] Specifically, different concentrations of E09 (0.01, 0.1, 1, 10, 100 μM) were added to purified recombinant HTRA1 and HTRA2, and the activity of HTRA1 and HTRA2 was 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. Graphs showing the fluorescence signals of HTRA1 and HTRA2 are shown in Figures 2 and 3.

[0105] Figure 2 is a graph showing the change in activity of HTRA1 according to the E09 dose: Figure 2(a) is a graph showing the change in activity of HTRA1 when the E09 dose is 1 nM to 1 μM, Figure 2(b) is a graph showing the change in activity of HTRA1 when the E09 dose is 1 nM to 10 μM, Figure 2(c) is a graph showing the change in activity 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 added (final concentration 10 μM) and when it is not added.

[0106] Figure 3 is a graph showing the change in activity of HTRA2 according to the E09 dose: Figure 3(a) is a graph showing the change in activity of HTRA2 when the E09 dose is 1 nM to 1 μM, and Figure 3(b) is a graph showing the change in activity of HTRA2 when the E09 dose is 1 nM to 10 μM.

[0107] As shown in Figures 2 and 3, it was confirmed that the activity of HTRA1 and HTRA2 increased as the dose of E09 increased. This means that E09 activates HTRA1 and HTRA2 in a concentration-dependent manner and also activates HTRA1 in human blood samples.

[0109] Example 3. Confirmation of HTRA1 activity depending on the presence or absence of E09 in HeLa cells

[0110] To determine 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 HTRA1 depending on the presence of E09.

[0111] Specifically, cells treated with an HTRA1 activity-based probe (DS-1-94) and E09 (HTRA activator) were placed in a 37°C incubator for 1 hour. Afterward, the cells were observed using a fluorescence microscope to determine how the probes reacted in living cells, and the results are shown in Figure 4.

[0112] Figure 4 is a fluorescence microscope image showing the activity of HTRA1 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).

[0113] As shown in Figure 4, it was confirmed that the fluorescence signal was significantly increased in cells treated with activator (E09). This means that when cells are treated with E09, the activity of HTRA1 increases.

[0115] Example 4. Measurement of change in HTRA activity following the addition of HTRA activator

[0116] 4.1 Preparation of HTRA activity-based probes

[0117] An HTRA activity-based probe was prepared using the method disclosed in the inventor's prior registered patent KR 10-2302971.

[0118] 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 is 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, to confirm subfamily selectivity, each series was designed by changing the P2, P3, and P4 positions of the P1-V tetrapeptide probe series and the P1-L tetrapeptide probe series to various types of amino acid functional groups. Thirteen types of tetrapeptide probes were synthesized based on the HTRA substrate amino acid sequences reported in previous studies.

[0119] 4.2 Comparison of HTRA Enzyme Activity by HTRA Agitator

[0120] HTRA activity was measured using the HTRA activity-based probe synthesized by the method of Example 4.1 and E09. Changes in HTRA1 activity were measured when caseine, known as a substrate of the HTRA enzyme, or PDZ-opt (GQYYFV), known as a peptide that activates the HTRA enzyme, was added, and when E09 was added.

[0121] Specifically, 2 μg of β-caseine or PDZ-opt (GQYYFV) or 10 μM of E09 was added to the HTRA1 enzyme by the method of Example 1, and after being left at room temperature for 15 minutes, HTRA1 activity was measured at 37°C for 3 hours by the same method as in Example 1, and the results are shown in Figure 5.

[0122] Figure 5 is a graph showing the enzyme activity of HTRA1 using caseine, known as a substrate of the HTRA enzyme, or PDZ-opt (GQYYFV) and E09, known as peptides that activate the HTRA enzyme.

[0123] As shown in Figure 5, it was confirmed that E09 activates the HTRA1 enzyme significantly better than caseine or PDZ-opt (GQYYFV) at room temperature.

[0124] These results indicate that using E09 allows for the effective measurement of HTRA activity even with low doses of HTRA enzyme, without temperature changes or the addition of substrates.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of age-related macular degeneration (AMD), comprising as an active ingredient a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are methyl. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A pharmaceutical composition according to claim 1, wherein the macular degeneration is selected from the group consisting of wet macular degeneration, dry macular degeneration, geographic atrophy, and diabetic retinopathy. Claim 6 delete Claim 7 A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition activates HTRA (High temperature requirement A). Claim 8 A pharmaceutical composition according to claim 7, wherein the HTRA is one or more selected from HTRA1 and HTRA2. Claim 9 A health functional food for the prevention or improvement of age-related macular degeneration (AMD), comprising as an active ingredient a compound of the following chemical formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.[Chemical Formula 1] In the above chemical formula 1, R1 and R2 are methyl. Claim 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 methyl. Claim 11 A reagent composition for HTRA activation according to claim 10, wherein the reagent composition comprises a compound of Formula 1 at a concentration of 10 μM to 100 μM. Claim 12 A method for screening HTRA activity modifiers, comprising: a step of activating HTRA in vitro by treating a sample containing HTRA (High temperature requirement A) with a compound of Formula 1, a stereoisomer thereof, a solvate thereof, a hydrate thereof, or an acceptable salt thereof; a step of treating the 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. [Formula 1] In the above chemical formula 1, R1 and R2 are methyl. Claim 13 A screening method for HTRA activity modifiers according to claim 12, wherein the comparing step includes the step of selecting a candidate substance that has changed the HTRA activity level as an HTRA modifier. Claim 14 A screening method for HTRA activity modifiers according to claim 12, wherein the sample is selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

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