Methods and compositions for the treatment of inflammation and inflammatory conditions

Abstract

Disclosed herein are methods of treating a patient having an inflammation or an inflammatory disease or a disease in which inflammation is present, such as cardiovascular inflammation or vascular endothelial inflammation, such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, stroke, sepsis, or a viral infection such as a coronavirus infection, such as a human patient having SARS-CoV-2. These methods comprise administering to the patient an effective amount of a compound described herein for treating the patient. Also provided are NCOA7 activating compounds and compositions. Also provided is a gene editing method for reducing inflammation or an inflammatory condition in a patient having C at SNP rs11154337, the method comprising editing C to G.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 346,453, filed May 27, 2022, and U.S. Provisional Patent Application No. 63 / 502,499, filed May 16, 2023, the disclosures of which are hereby incorporated by reference in their entirety into this specification.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under grant number HL138437 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The Sequence Listing associated with this application was filed electronically via the Patent Center and is hereby incorporated by reference in its entirety into this specification. The name of the XML file containing the Sequence Listing is 2302529.xml. The size of the XML file is 31,855 bytes, and the XML file was created on May 25, 2023.

[0004] Inflammation is a crucial component of a vast number of diseases, including vascular diseases and heart diseases, as well as non - vascular diseases such as sepsis, COVID (Coronavirus Disease), ARDS (Acute Respiratory Distress Syndrome), acute lung injury, stroke, neurodegeneration, cancer, and autoimmune diseases. In the example of vascular inflammation, inflammation regulates major vascular and endothelial pathologies (such as atherosclerosis, essential hypertension, peripheral vascular disease, and restenosis), but in particular, in pulmonary vascular diseases such as pulmonary arterial hypertension (PAH) or other subtypes of pulmonary hypertension (groups 2 - 5), the exact causal mechanisms remain unclear. In one example, PAH includes diseases such as idiopathic PAH, hereditary PAH (e.g., BMPR - 2, etc.), connective tissue disorder - related PAH, HIV infection, portopulmonary hypertension, congenital heart disease, schistosomiasis, and hemolytic anemia, pulmonary veno - occlusive disease, and persistent pulmonary hypertension of the newborn as a class.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Identification of an active pharmaceutical ingredient capable of reducing inflammation, for example, inflammation of blood vessels or vascular endothelium, such as a drug, is useful for treating various diseases, such as inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (for example, diseases having or presenting vascular endothelial inflammation, such as pulmonary hypertension (including groups 1 to 5 types of PH, such as pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease, pulmonary hypertension due to chronic thrombus in the lung, and pulmonary hypertension of unknown origin), restenosis, essential hypertension, atherosclerosis, and stroke), diseases characterized by vascular inflammation, or diseases of innate and acquired immunity, such as autoimmune diseases, heart diseases, lung diseases, sepsis, cancer, and neurodegeneration, for example, treatment of inflammation associated with heart diseases, lung diseases, sepsis, cancer, and neurodegeneration. Identification of such useful active pharmaceutical ingredients and related methods of use are desired.

Means for Solving the Problems

[0006] Overview A method for treating inflammation in a patient such as a human patient is provided. This method involves administering to the patient a structure:

Chemical formula

[0007] A method for treating pulmonary arterial hypertension (PAH) in a patient, comprising administering to said patient a structure:

Chemical formula

[0008] Structure: [Chemical formula] [In the formula, R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, X3 are independently N or C; X4 is ortho, meta or para to X1 and is N or C; Y2 is -H, -C 1-3 alkyl, halo,, or -NO2; Y1 is -H, -C 1-3 alkyl, halo, -NO2, -CN, -CF3, -SO2R4 (wherein R4 is -OH, -C 1-3 alkyl), -NHR5 (wherein R5 is H or -C 1-3 alkyl), -NHR6 (wherein R6 is -H or C 1-3 alkyl), -NHC(O)-R7 (wherein R7 is -H or -C 1-3is alkyl), -OR8 (wherein R8 is, -H or -C 1-3 is alkyl), -OC(O)-R9 (wherein R9 is, -H or -C 1-3 is alkyl), -C(O)-R 10 (wherein R 10 is, -H or -C 1-3 is alkyl), or -C(O)-R 11 -R 12 (wherein R 11 is O or NH, and R 12 is -H, -C 1-3 is alkyl)] A compound comprising the same, or a pharmaceutically acceptable salt thereof (excluding MolPort-004-267-958) is provided.

[0009] Structure:

Chemical formula

[0010] Also provided is a method for treating a patient having C at SNP rs11154337 and having inflammation and / or an inflammatory disease, such as cardiovascular inflammation or vascular inflammation, or associated with a disease such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, viral infection, bacterial infection, fungal infection, parasitic infection, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegeneration, cancer, autoimmune disease, or innate and acquired immune diseases, the method comprising changing one or more C's of SNP rs11154337 to G using gene editing such as, for example, but not limited to, methods based on CRISPR / Cas9 or TALEN.

[0011] The following shows non-limiting aspects, embodiments, and examples as examples of the present invention. Item 1. A method for treating inflammation in a patient such as a human patient, comprising administering to the patient a structure:

Chemical formula

[0012] Item 2. The method according to Item 1, wherein the inflammation is cardiovascular inflammation or vascular inflammation, or is associated with a disease such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, viral infection, bacterial infection, fungal infection, parasitic infection, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegeneration, cancer, autoimmune disease, or innate and acquired immune diseases.

[0013] Item 3. The method according to Item 1, wherein the patient has vascular inflammation.

[0014] Item 4. The method according to Item 1, wherein the inflammation is associated with a viral infection or a bacterial infection.

[0015] Item 5. The method according to Item 2, wherein the inflammation is associated with the patient's coronavirus infection, optionally with severe acute respiratory syndrome from the patient's coronavirus infection.

[0016] Item 6. The method according to Item 2, wherein the inflammation is associated with the patient's bacterial infection.

[0017] Item 7. The method according to Item 6, wherein the bacterial infection is the patient's Klebsiella pneumoniae infection, and optionally reduces or prevents the patient's lung injury.

[0018] Item 8. The method according to Item 1, wherein the inflammation is associated with one or more of pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease, pulmonary hypertension due to chronic thrombus in the lung, and pulmonary hypertension of unknown cause.

[0019] Item 9. A method for treating pulmonary arterial hypertension (PAH) in a patient, comprising administering to the patient a structure:

Chemical Formula

[0020] The method according to any one of claims 1 to 9, wherein Z is NH in claim 10.

[0021] The method according to any one of claims 1 to 10, wherein R1 and R2 are independently Me or H in claim 11.

[0022] The method according to any one of claims 1 to 10, wherein R1 and R2 are H in claim 12.

[0023] The method according to any one of claims 1 to 9, wherein the compound is compound 958 (MolPort-004-267-958) or a pharmaceutically acceptable salt thereof in claim 13.

[0024] The method according to claim 14, wherein the compound has the exemplary structure:

Chemical formula

[0025] Claim 15 The method according to any one of claims 1 to 14, wherein an effective amount of a compound or a pharmaceutically acceptable salt thereof that reduces the patient's inflammation or treats the patient's pulmonary hypertension is administered to the patient.

[0026] Claim 16 The method according to any one of claims 1 to 14, comprising administering to the patient from 1 μg to 10 g, or from 1 ng to 100 mg / kg of the compound, or to a concentration in the range of 1 to 40 μM in a patient's body fluid such as blood, serum, plasma, etc.

[0027] Claim 17 The method according to any one of claims 1 to 16, wherein the patient is heterozygous or homozygous for C of rs11154337.

[0028] Claim 18 The method according to any one of claims 1 to 16, wherein the patient is homozygous for C of rs11154337.

[0029] Claim 19 The method according to claim 17 or 18, further comprising obtaining the patient's genetic data and determining whether the patient has one or two alleles with respect to C of rs11154337.

[0030] Claim 20 The method according to claim 17 or 18, further comprising determining whether the patient has one or two alleles with respect to C of rs11154337.

[0031] Claim 21 The method according to claim 5, wherein the coronavirus infection is one or more of diseases caused thereby such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), or Coronavirus Disease 2019 (COVID-19).

[0032] Item 22. The method according to item 21, wherein the coronavirus is SARS-CoV-2.

[0033] Item 23. The method according to item 1, for reducing the infectivity of coronavirus infection or herpesvirus infection in cells.

[0034] Item 24. Structure: [Chemical formula] [In the formula, R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, X3 are independently N or C; X4 is ortho, meta or para to X1 and is N or C; Y2 is -H, -C 1-3 alkyl, halo,, or -NO2; Y1 is -H, -C 1-3 alkyl, halo, -NO2, -CN, -CF3, -SO2R4 (where R4 is -OH, -C 1-3 alkyl), -NHR5 (where R5 is H or -C 1-3 alkyl), -NHR6 (where R6 is -H or C 1-3 alkyl), -NHC(O)-R7 (where R7 is -H or -C 1-3 alkyl), -OR8 (where R8 is -H or -C 1-3 alkyl), -OC(O)-R9 (where R9 is -H or -C 1-3 alkyl), -C(O)-R 10 (where R 10 is -H or -C 1-3 alkyl), or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 is -H, -C 1-3 alkyl)] A compound comprising the same, or a pharmaceutically acceptable salt thereof (excluding MolPort-004-267-958).

[0035] The compound according to item 24, wherein Z is NH.

[0036] The compound according to item 24 or 25, wherein R1 and R2 are independently Me or H.

[0037] The compound according to item 24 or 25, wherein R1 and R2 are H.

[0038] Item 28 Structure:

Chemical formula

[0039] Item 29 Structure:

Chemical formula

[0040] The composition according to item 29, wherein Z is NH as described in item 30.

[0041] The composition according to item 29 or 30, wherein R1 and R2 are independently Me or H as described in item 31.

[0042] The composition according to item 29 or 30, wherein R1 and R2 are H as described in item 32.

[0043] Item 33 Structure:

Chemical formula

[0044] The composition according to item 29, wherein the compound is MolPort-004-267-958.

[0045] A method for treating a patient having a C at SNP rs11154337 and having inflammation and / or an inflammatory disease, such as cardiovascular inflammation or vascular inflammation, or associated with a disease such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, viral infection, bacterial infection, fungal infection, parasitic infection, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegeneration, cancer, autoimmune disease, or innate and acquired immune diseases, the method comprising changing one or more Cs of SNP rs11154337 to G using gene editing such as, but not limited to, a method based on CRISPR / Cas9 or TALEN.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0047] Detailed Description The following description is merely exemplary in nature and is not intended to limit the invention, its application, or uses in any way. This specification is provided to enable those skilled in the art to make and use the invention, and specific examples are shown for that purpose, but these should not be considered limiting. It will be apparent to those skilled in the art that various modifications as set forth below will be included within the scope of the appended claims. The invention should not be considered limited to the presently disclosed aspects, whether or not such aspects are provided in an example or elsewhere in this specification.

[0048] The use of numerical values within the various ranges specified in this application is, unless otherwise expressly indicated, described as an approximation as if the word “about” preceded both the minimum and maximum values within the recited range. Thus, minor variations above and below the recited ranges can be used to achieve substantially the same results as the values within the range. Also, unless otherwise indicated, the disclosure of these ranges is intended as a continuous range that includes any value between the minimum and maximum values. For the definitions provided in this specification, those definitions refer to the word form, synonyms, and grammatical variations of those words or phrases. As used in this specification, “a” and “an” refer to one or more. Patent publications cited below are incorporated by reference in their entirety within the scope of their technical disclosure and consistency with this specification.

[0049] As used in this specification, the terms “comprising,” “comprise,” or “comprised” and their variations are open-ended and do not exclude the presence of other elements not specified. In contrast, the term “consisting of” and its variations are intended to be closed-ended and exclude any additional elements other than trace amounts.

[0050] As used herein, the terms "patient" or "subject" refer to those belonging to the animal kingdom including, but not limited to, humans, and "mammal" refers to all mammals including, but not limited to, humans.

[0051] As used herein, "treatment" of inflammation, cardiovascular inflammation, vascular inflammation (e.g., vascular inflammation (e.g., endothelial inflammation), diseases characterized by vascular inflammation, or innate and acquired immune diseases) means administration to a patient by any suitable dosing schedule, procedure, and / or route of administration of a composition, device, or construct for the purpose of achieving a desired clinical / medical endpoint, which includes, but is not limited to, in the case of PAH, a mean pulmonary artery pressure ≧ 25 mmHg measured by right heart catheterization in the supine position at rest. Reduction or prevention of further development of vascular endothelial inflammation, e.g., PAH. An amount effective to treat a patient, administered by any suitable route of any reagent or therapeutic agent, is an amount capable of preventing, reducing, and / or eliminating endothelial inflammation such as PAH, and / or reducing the severity of one or more symptoms of endothelial inflammation such as PAH, e.g., an amount such that the mean pulmonary artery pressure measured by right heart catheterization in the supine position at rest is < 25 mmHg. The therapeutically effective amount of each therapeutic agent may range from 1 pg to 10 g per dose, and includes, for example, but is not limited to, any amount therebetween such as 1 ng, 1 μg, 1 mg, 10 mg, 100 mg, or 1 g per dose. The therapeutic agent can be administered by any effective route, e.g., as a single dose or bolus, at regular or irregular intervals, in amounts and at intervals directed by any clinical parameter of the patient, or continuously.

[0052] Cardiovascular inflammations, such as vascular inflammation (e.g., endothelial inflammation or vasculitis of the vascular endothelium), are associated with not only PAH but also, without limitation, vasculitis including peripheral arterial disease, large vessel vasculitis, medium vessel vasculitis, and small vessel vasculitis, infectious diseases, chronic vascular inflammatory diseases such as atherosclerosis, inflammatory or inflammation-related conditions such as restenosis, essential hypertension, stroke, and other diseases (e.g., see McLaughlin VV, McGoon MD. Pulmonary arterial hypertension. Circulation. 2006 Sep 26;114(13):1417-31). Other diseases that can be effectively treated by the methods described herein include innate and acquired immune diseases such as heart failure (HFpEF and HFrEF), myocarditis, and atrial fibrillation, and inflammatory myeloid cells also appear to exacerbate symptoms and disease severity. Pulmonary hypertension, including PAH, pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease, pulmonary hypertension due to chronic thromboembolism of the lung, and pulmonary hypertension of unknown origin, can be effectively treated by the compositions and methods described herein. Other innate and acquired immune diseases include, for example, inflammation associated with heart disease, lung disease, sepsis, cancer, and neurodegeneration, such as heart disease, lung disease, sepsis, cancer, and neurodegeneration.

[0053] As used herein, "treatment" of a coronavirus infection, without limitation, means administration to a patient by any suitable dosing schedule, procedure, and / or route of administration of a composition, device, or construct for the purpose of achieving a desirable clinical / medical endpoint, including, for example, reducing or preventing further development of a coronavirus infection, as determined below. An effective amount, administered by any suitable route, of any reagent or therapeutic agent to treat a patient is an amount that can prevent, reduce, and / or eliminate a coronavirus infection and / or reduce the severity of one or more symptoms of a coronavirus infection, such as fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, loss of taste or smell, sore throat, nasal congestion or runny nose, nausea or vomiting, or diarrhea. The therapeutically effective amount of each therapeutic agent, such as the compounds (1) exemplified below in ami , may range from 1 pg per dose to 10 g per dose and may include any amount therebetween, for example, without limitation, 1 ng, 1 μg, 1 mg, 10 mg, 100 mg, or 1 g per dose. The therapeutic agent may be administered by any effective route, for example, as a single dose or bolus, at regular or irregular intervals, in amounts and at intervals directed by any clinical parameter of the patient, or continuously. ami The therapeutically effective amount of the compound (1) described below and exemplified in ami may range from 1 pg per dose to 10 g per dose and may include any amount therebetween, for example, without limitation, 1 ng, 1 μg, 1 mg, 10 mg, 100 mg, or 1 g per dose. The therapeutic agent may be administered by any effective route, for example, as a single dose or bolus, at regular or irregular intervals, in amounts and at intervals directed by any clinical parameter of the patient, or continuously.

[0054] 958 and 958 amiThe active ingredients such as the compound (1) described below, exemplified by, can be formulated into a composition suitable for use in a pharmaceutical dosage form or a pharmaceutical product having the compound as an active ingredient or can be produced by other methods. The composition may comprise a pharmaceutically acceptable carrier or excipient. An excipient is an inert substance used as a carrier for the active ingredient of a medicine. Although "inert", an excipient can facilitate and assist in enhancing the delivery or bioavailability of the active ingredient in a pharmaceutical product. Non-limiting examples of useful excipients include antiadhesives, binders, rheology modifiers, coating agents, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, fragrances, colorants, flow promoters, lubricants, preservatives, antioxidants, adsorbents, vitamins, sweeteners, etc., such as those available in the pharmaceutical / formulation art.

[0055] Useful dosage forms include, for example, but are not limited to, intravenous, intramuscular, intraocular, or intraperitoneal solutions, oral tablets or liquids, topical ointments or creams, and transdermal devices (e.g., patches). In one embodiment, the compound is a sterile solution comprising an active ingredient (drug or compound) and a solvent such as water, physiological saline, lactated Ringer's solution, or phosphate buffered saline (PBS). Further excipients such as polyethylene glycol, emulsifiers, salts, and buffers may be included in the solution.

[0056] Suitable dosage forms may include single-dose or multi-dose vials containing a composition comprising an active ingredient useful for the treatment of coronavirus infection as described herein, or other containers such as medical syringes or droppers.

[0057] Pharmaceutical formulations suitable for administration include aqueous and non-aqueous sterile solutions, which may contain, in addition to the active pharmaceutical ingredient or drug, for example, but not limited to, antioxidants, buffers, bacteriostatic agents, lipids, liposomes, lipid nanoparticles, emulsifiers, suspending agents, and rheology modifiers. The formulations can be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state that requires only the addition of a sterile liquid carrier, such as water for injection, immediately before use. It is also possible to prepare immediate-use solutions and suspensions from sterile powders, granules, and tablets.

[0058] Therapeutic compositions are generally required to be sterile and stable under the conditions of manufacture and storage. For example, a sterile injectable solution can be prepared by incorporating the required amount of the active agent in a suitable solvent containing one or a combination of the ingredients listed herein, if necessary, and then filtering and sterilizing. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient and the desired additional ingredients from a previously filtered and sterilized solution. The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as monostearates and gelatin.

[0059] "A therapeutically effective amount" refers to the amount of a pharmaceutical or active agent that is effective at dosages and for periods of time necessary to achieve the desired therapeutic result. For the treatment of a particular condition, an "effective amount" is the amount of an active agent or dosage form, such as a single or multiple doses, that is effective in achieving a determinable endpoint. An "effective amount" is preferably safe - at least to the extent that the benefits of treatment outweigh the disadvantages and / or to the extent that the disadvantages are acceptable to a person of ordinary skill in the art and / or to an appropriate regulatory authority such as the United States Food and Drug Administration. The therapeutically effective amount of an active agent can vary depending on factors such as the condition of the individual, age, gender, and weight, as well as the ability of the active agent to elicit the desired response in the individual. "A prophylactically effective amount" refers to the amount that is effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Generally, since prophylactic dosages are used in subjects at a pre-disease or early stage of the disease, a prophylactically effective amount may be less than a therapeutically effective amount.

[0060] The dosing regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single dose or bolus may be administered, doses divided over several times may be administered over time, or the composition may be administered continuously or pulsatile, with doses or sub-doses being administered at regular intervals, such as every 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes, every 2 - 12 hours per day, or every other day, and may be proportionally decreased or increased as indicated by the exigencies of the treatment situation. In some cases, it may be particularly advantageous to formulate the composition in unit dosage form to facilitate administration and to uniformize the dosage. The specifications of the unit dosage form are determined by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of hypersensitivity in an individual.

[0061] An effective amount for treating a patient's inflammation or inflammatory condition is from 1 μg to 10 g per day, or 1 ng to 100 mg / kg of Compound 958. For example, the compound can be in an amount of 20 μM ± 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 0.5%, 1%, for example 1 to 40 μM, or any increment therebetween, in the patient's body fluids such as blood, serum, plasma, etc.

[0062] Virus-related inflammations, such as respiratory inflammations, can also be treated with the compounds described herein. For example, coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, coronaviruses cause respiratory infections that can range from mild to lethal. Mild diseases in humans include some cases of the common cold (which can also be caused by other viruses, mainly rhinoviruses), while more lethal species can cause Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), or Coronavirus Disease 2019 (COVID-19). In cows and pigs, coronaviruses cause diarrhea, and in mice, they cause hepatitis and encephalomyelitis.

[0063] SARS-CoV-2 is the virus that causes COVID-19, a respiratory disease that is the cause of the COVID-19 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA virus that infects humans. The diameter of each SARS-CoV-2 virion is approximately 50-200 nanometers. SARS-CoV-2 has four structural proteins known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins, with the N protein holding the RNA genome and the S, E, and M proteins together forming the viral envelope. The spike protein is the protein for the virus to attach to and fuse with the host cell membrane. Specifically, its S1 subunit mediates attachment and its S2 subunit catalyzes fusion. Symptoms of SARS-CoV-2 infection can appear 2-14 days after virus exposure. Symptoms of SARS-CoV-2 include, but are not limited to, fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, or diarrhea.

[0064] As shown below, compounds 958 and 958 amiis shown to be useful for the treatment of inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., having vascular endothelial inflammation, or diseases having vascular endothelial inflammation as a symptom such as pulmonary arterial hypertension, restenosis, essential hypertension, atherosclerosis, and stroke), diseases characterized by vascular inflammation, or innate and acquired immune diseases such as heart diseases, lung diseases, sepsis, cancer, and neurodegeneration, e.g., inflammation associated with heart diseases, lung diseases, sepsis, cancer, and neurodegeneration. This compound can be used for treating viral infections, e.g., inflammation associated with viral infections such as SARS-CoV-2 or other SARS or MERS virus infections. Compound 958, e.g., but not limited to, MolPort-004-267-958, can be described as (6,7-dihydroxy-2-oxo-2H-chromen-4-yl)methyl 4-oxo-3-phenyl-3,4-dihydrophthalazine-1-carboxylate, structure: [Chemical formula] has it, includes its pharmaceutically acceptable salts, and may include its equivalent derivative compounds, e.g., esters or prodrugs.

[0065] Compound 958 ami has the structure: [Chemical formula] has it.

[0066] Compound 958 and 958 ami has the following general structure where equivalent activity is expected: [Chemical formula] [In the formula, both or each of R1 and R2 (independently) is -H or -C 1-3 alkyl; Z is O or NH; X1, X2, X3 are independently N or C; X4 is ortho, meta, or para to X1 and is N or C; Y2 is -H, -C 1-3 alkyl, halo (-F, -Cl, -Br, or -I), or -NO2 (nitro); Y1 is -H, -C 1-3 alkyl, halo (-F, -Cl, -Br, or -I), -NO2, -CN (nitrile), -CF3 (trifluoromethyl), -SO2R4 (where R4 is -OH, -C 1-3 alkyl), -NHR5 (where R5 is H, -C 1-3 alkyl), -NHR6 (where R6 is -H, -C 1-3 alkyl), -NHC(O)-R7 (where R7 is -H, -C 1-3 alkyl), -OR8 (where R8 is -H, -C 1-3 alkyl), -OC(O)-R9 (where R9 is -H, -C 1-3 alkyl, or C 1-3 alkoxy), -C(O)-R 10 (where R 10 is -H, -C 1-3 alkyl), or -C(O)-R 11 -R 12 (R 11 can be O or NH; R 12 is -H, -C 1-3 alkyl)), which is an example of compound (1) provided herein, including its pharmaceutically acceptable salts, and may include its equivalent derivative compounds, such as esters or prodrugs. When the "R group" is an ester or an amide, the ester or amide can form a bond connecting the active moiety to another moiety such as an inactive moiety or a carrier. As used herein, "moiety" refers to a part of a molecule such as a part to which activity or functionality can be ascribed.

[0067] As used herein, "alkyl" refers to, for example, a straight-chain, branched-chain, or cyclic hydrocarbon (hydrocarbyl) group containing, for example, from 1 to about 24 carbon atoms, such as, but not limited to, C containing 1, 2, or 3 carbons​1-3 A group, for example, methyl, ethyl, or propyl is included. "Alkoxy" refers to an alkyl group bonded through oxygen, for example, methoxy (-OCH3), ethoxy (-O-CH2-CH3), or propyloxy (for example, -O-CH2-CH2-CH3 or -O-CH(CH3)2), and is collectively referred to as C 1-3 alkoxy. "Halogen", "halide", and "halo" refer to -F, -CI, -Br, and / or -I. "Alkylene" and "substituted alkylene" refer to divalent alkyl and divalent substituted alkyl, respectively, and include, but are not limited to, ethylene (-CH2-CH2-).

[0068] Compound (1) is compound 958 and 958 ami As described above with respect to compounds 958 and 958, in the treatment of patients, for example, in inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (for example, having vascular endothelial inflammation, or diseases such as pulmonary arterial hypertension, restenosis, essential hypertension, atherosclerosis, and stroke that have vascular endothelial inflammation as a symptom), diseases characterized by vascular inflammation, or autoimmune diseases, natural and acquired immune diseases such as heart diseases, lung diseases, sepsis, cancer, and neurodegeneration, for example, in the treatment of inflammation associated with heart diseases, lung diseases, sepsis, cancer, and neurodegeneration. This compound can be used to treat viral infections such as SARS-CoV-2 or other SARS or MERS virus infections, for example, inflammation associated with viral infections.

[0069] The therapeutically effective amount of each compound for the treatment of a disease as described herein may range from 1 pg per dose to 10 g per dose, and includes, but is not limited to, 1 ng, 1 μg, 1 mg, 10 mg, 100 mg, or 1 g per dose to the patient, for example, 10 ng / kg / day to 1 g / kg / day, or 1 μg / kg / day to 100 mg / kg / day, or increments therebetween. The therapeutically effective amount may range from 1 μg to 10 g per day, or in the range of 1 ng to 100 mg / kg of Compound 958, or an equivalent amount of another compound described herein, or, for example, 20 μM ± 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or any increment therebetween in a patient's body fluid such as blood, serum, plasma. The therapeutic agent may be administered to the patient by any effective route, for example, as a single dose or bolus, at regular or irregular intervals, or continuously, in an amount and at intervals directed by any clinical parameter of the patient.

[0070] As shown below, additional compounds are evaluated to be useful for the treatment of inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., having vascular endothelial inflammation, or diseases having vascular endothelial inflammation as a symptom such as pulmonary arterial hypertension, restenosis, essential hypertension, atherosclerosis, and stroke), diseases characterized by vascular inflammation, or innate and acquired immune diseases such as heart disease, lung disease, sepsis, cancer, and neurodegeneration, for example, inflammation associated with heart disease, lung disease, sepsis, cancer, and neurodegeneration. The present compounds can be used to treat viral infections such as SARS-CoV-2 or other SARS or MERS virus infections, for example, inflammation associated with viral infection. The compounds listed in FIGS. 11, 16A - 16E can be provided as their pharmaceutically acceptable salts and may include equivalent derivative compounds such as esters or prodrugs thereof.

[0071] The single nucleotide polymorphism (SNP) rs11154337 is a polymorphism within the NCOA7 gene. Figures 1 and 2 show the sequence of rs11154337 and its position within the intron of the NCOA7 gene (see also US Patent Publication No. 2021 / 0309998A1, the disclosure of which is incorporated herein by reference in its entirety). Figures 3A and 3B provide exemplary NCOA7 mRNA (cDNA) sequences.

[0072] NCOA7 is the protein product of the NCOA7 gene. An exemplary NCOA7 mRNA sequence (SEQ ID NO: 2) is shown in FIGS. 3A and 3B. The NCOA7 gene has a candidate SNP called rs11154337 (SEQ ID NO: 1). An exemplary rs11154337 sequence is shown in FIGS. 1 and 2. rs11154337 is located in the promoter of the interferon-inducible isoform of NCOA7 (NCOA7short), which was first identified by the inventors in an unpublished genome-wide association study (GWAS) regarding survival in human pulmonary arterial hypertension. Other mutations or polymorphisms located in the same intron as rs11154337, as shown in FIG. 2 for example, or in linkage disequilibrium with rs11154337, may exhibit a high-risk genotype or functionally affect the expression of NCOA7, and thus, similar to or in combination with rs11154337, may be useful for detecting individuals particularly susceptible to coronavirus infection. For example, correcting the risk polymorphism by gene editing of functional polymorphisms that affect the expression of NCOA7 may reduce the infectivity of the coronavirus. The SNP rs11154337 (SEQ ID NO: 1) is present in an intron region where both the RelA / p65 subunit of NF-kB and STAT1 are predicted to bind. From the perspective of antibacterial defense, this duality suggests a functional cooperation between two host defense pathways: (1) initial detection at the cell membrane via Toll-like receptors and the NF-kB pathway, and (2) potential pathogen escape in endosomes that triggers interferon-mediated responses and STAT1 / 2 activation via Janus tyrosine kinase (JAK). Thus, the inventors' molecular studies defined NCOA7 as an upregulatory factor in ECs in response to inflammatory cytokines, and further, both inhibition of STAT1 / 2 signaling via the JAK inhibitor momelotinib and RNAi of RelA / p65 suppressed the upregulation of NCOA7 via IL-1β. The inventors demonstrated that NCOA7 regulates endothelial immune activation and subsequent leukocyte adhesion and perhaps infiltration. To that end, NCOA7 alters lysosomal acidification, a feature that has been uniquely found to affect the entry of other enveloped viruses such as influenza.Furthermore, using an in vitro biochemical assay of the binding of nuclear proteins to induced pluripotent stem cells (iPSCs) of an isogenic line edited with SNP rs11154337 and CRISPR-Cas9, allele-specific binding to the NF-kB subunit RelA / p65 that promotes allele-specific expression of NCOA7 was found.

[0073] Lysosomes are membrane-bound organelles found in many animal cells. Lysosomes are spherical vesicles containing hydrolytic enzymes that can break down many types of biomolecules. Lysosomes have a specific composition both in their membrane proteins and luminal proteins. The pH of the lumen (about 4.5 - 5.0) is optimal for the enzymes involved in hydrolysis and is similar to the activity of the stomach. In addition to the breakdown of polymers, lysosomes are involved in various cellular processes such as secretion, cell membrane repair, apoptosis, cell signaling, and energy metabolism. Lysosomes function as the cell's waste disposal system by digesting used substances within the cytoplasm both inside and outside the cell. Substances from outside the cell are taken up by endocytosis, and substances from inside the cell are digested by autophagy. The size of lysosomes varies from 0.1 μm to 1.2 μm. The pH of lysosomes is about 4.5 - 5.0, so the interior of lysosomes is acidic compared to the slightly basic cytosol (pH 7.2). Lysosomes maintain this pH difference by pumping protons (H + ions) across the membrane from the cytoplasm through proton pumps and chloride ion channels. The transport of protons is carried out by vacuolar ATPase, and the countertransport of chloride ions is carried out by the ClC-7 Cl - / H + antiporter.

[0074] The research of the present inventors has established a paradigm that links lysosome biology and oxysterol and bile acid metabolism to endothelial inflammation, providing broad implications for precision medicine approaches in pulmonary arterial hypertension. The present inventors have also found evidence of a genetic association between this NCOA7 SNP and essential hypertension, atherosclerosis, and stroke, revealing that this target is also associated with these other vascular diseases. In pilot data, the present inventors have found an association between these same oxysterols and bile acids in sepsis, suggesting that targeting NCOA7 may be beneficial not only in this severe disease but also in diseases of both acquired and innate immunity in general.

[0075] In this process, the present inventors used a computational prediction approach to identify specific small molecules that may activate or inhibit NCOA7, based on the prediction that they are likely to bind to the enzymatic pocket of the Tre2 / Bub2 / Cdc16 (TBC), lysine motif (LysM), domain catalytic (TLDc) domains of NCOA7. One of the top hits, compound 958, (e.g., (6,7-dihydroxy-2-oxo-2H-chromen-4-yl)methyl 4-oxo-3-phenyl-3,4-dihydrophthalazine-1-carboxylate), was found to have predicted activity to change the pH of lysosomes and downregulate cholesterol metabolism as well as inflammatory activity in endothelial cells. Also, those compounds may exhibit such therapeutic activity in other non-vascular diseases of both acquired and innate immunity. Finally, as supported by the present inventors' pilot data showing that those compounds can inhibit the entry of SARS-CoV-2 pseudotyped virus into human cells, they may serve as a new therapy for COVID-19.

[0076] In aspects and embodiments 1. The present invention is based on novel and unpublished mechanistic data that describe the role of NCOA7 in the control of lysosomal activity, sterol homeostasis, and inflammation. 2. In particular, the present invention aims at novel pathways and target sets that have not been attempted heretofore in cardiovascular diseases, sepsis, or COVID-19. 3. Importantly, the associations of NCOA7 and the target pathways with human pulmonary arterial hypertension, essential hypertension, stroke, and atherosclerosis are based on population-level human gene and metabolome data. 4. The molecule (6,7-dihydroxy-2-oxo-2H-chromen-4-yl)methyl 4-oxo-3-phenyl-3,4-dihydrophthalazine-1-carboxylate has no known protein targets and has not been studied with respect to human diseases or inflammation in general. 5. The computational processes by which these small molecule compounds were predicted have not been applied to NCOA7 or the TLDc domain in the past.

[0077] In a further embodiment, provided is a method of treating a patient having C at SNP rs11154337 and having inflammation and / or an inflammatory disease, such as cardiovascular inflammation, vascular inflammation (e.g., having vascular endothelial inflammation, or a disease having vascular endothelial inflammation as a symptom such as pulmonary arterial hypertension, restenosis, essential hypertension, atherosclerosis, and stroke), a disease characterized by vascular inflammation, or a innate and adaptive immune disease, such as an autoimmune disease, heart disease, lung disease, sepsis, cancer, and neurodegeneration, e.g., inflammation associated with heart disease, lung disease, sepsis, cancer, and neurodegeneration. The method comprises the use of gene editing, such as, but not limited to, gene editing methods based on CRISPR / Cas9 or TALEN, to change one or more C's of SNP rs11154337 to G (for CRISPR / CAS9 technology and tools useful for the implementation of such technology, see, e.g., Abdelnour SA, Xie L, Hassanin AA, Zuo E, Lu Y. The Potential of CRISPR / Cas9 Gene Editing as a Treatment Strategy for Inherited Diseases. Front Cell Dev Biol. 2021 Dec 15;9:699597 and Chiao-Lin Chen, Jonathan Rodiger, Verena Chung, Raghuvir Viswanatha, Stephanie E Mohr, Yanhui Hu, Norbert Perrimon, SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing, G3 Genes|Genomes|Genetics, Volume 10, Issue 2, 1 February 2020, Pages 489-494, and also refer to commercial services for gene editing such as using the technology and expertise provided by CRISPR Therapeutics of Cambridge, Massachusetts).

Example

[0078] By utilizing large-scale and multi-dimensional metabolome and genome data together with in vitro and in vivo mechanism experiments, it has been found that NCOA7 controls lysosomal activity and endothelial sterol homeostasis and acts as a homeostatic brake to suppress oxysterol- and 7HOCA-induced inflammation, endothelial dysfunction, and PAH. Furthermore, when the G allele of the NCOA7 intron SNP rs11154337 is present, NCOA7 is increased, inflammation in PAH is reduced, and genetic evidence underlying the genetic association of SNP rs11154337 with PAH disease severity, the metabolomic association of oxysterols and bile acid signatures with PAH severity, and the genetic association of SNP rs11154337 with 7HOCA plasma levels is provided. Identification of active pharmaceutical ingredients, such as drugs, that can reduce inflammation, such as endothelial inflammation, provides therapeutic agents useful for the treatment of various inflammation-related diseases, including cardiovascular inflammation, vascular inflammation (e.g., diseases having inflammation of the vascular endothelium or diseases having vascular endothelial inflammation as a symptom, such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, and stroke), diseases characterized by vascular inflammation, or innate and acquired immune diseases. The following examples are illustrative of the claimed invention and demonstrate the concept.

[0079] Example 1 Vascular inflammation critically regulates the pathology of endothelial cells (ECs), but its causal mechanisms are not yet fully defined, particularly in pulmonary arterial hypertension (PAH). Although dysregulation of immunity and metabolic reprogramming are recognized as important elements of PAH pathology, a unified theory linking the two has not been established. In human pulmonary artery ECs, induction of nuclear receptor coactivator 7 (NCOA7), a gene previously identified as upregulated by PAM, promoted lysosomal acidification and suppressed EC immune activation, thereby inhibiting the production of inflammatory sterols. Conversely, a decrease in NCOA7 promoted lysosomal dysfunction and the production of inflammatory sterols and bile acids that promoted an EC phenotype consistent with PAH. In vivo, mice deficient in NCOA7 or treated externally with 7α-hydroxy-3-oxo-4-cholestenic acid (7HOCA), a representative metabolite of the NCOA7-dependent sterol signature, showed exacerbation of PAH. Given the clinical importance of this mechanism in controlling disease severity, an unbiased metabolome-wide association study of a multi-site PAH Biobank cohort (N = 2,796) identified a plasma signature containing the same NCOA7-dependent sterols and bile acids associated with PAH mortality (adjusted P < 1.1×10 -6)。Since the genetic predisposition of NCOA7 deficiency was shown to be widespread, the common variant intronic SNP rs11154337 in NCOA7 was found to control NCOA7 levels, lysosomal activity, sterol and bile acid production, and EC immune activation in isogenic iPSCs-derived ECs edited with the SNP. Correspondingly, in an independent single-center PAH cohort (N = 93), SNP rs11154337 was associated with PAH severity as shown by 6-minute walk distance (P = 0.0130, β = 58.15, 95% CI [14.45 - 119.36]) and mortality (P = 0.0250, hazard ratio = 0.44, 95% CI [0.21 - 0.90]). As a second validation, in a multi-center PAH cohort (N = 630), SNP rs11154337 was further associated with mortality (P = 0.0002; hazard ratio = 0.49, 95% CI [0.34 - 0.71]). Finally, using computational modeling and simulation of the functional domains of NCOA7, a novel activator of NCOA7 that prevents endothelial immune activation and reverses PAH markers was predicted and synthesized in monocrotaline rats. Taken together, this study established a genetic-metabolic paradigm linking lysosomal biology and sterol and bile acid processes to EC inflammation and proposed a novel therapy. This paradigm has broad implications not only for the molecular diagnosis and treatment of PAH but also for other vascular disorders that rely on the regulation of adaptive and innate immunity.

[0080] Vascular inflammation critically regulates the behavior of endothelial cells (ECs) in vascular diseases such as atherosclerosis, hypertension, stroke, and sepsis. In the lung, endothelial inflammation is a prominent feature of acute lung injury, processes mediated by pathogens such as SARS-CoV-2 infection, and pulmonary arterial hypertension (PAH) - a fatal and enigmatic vascular disease characterized by complex vascular remodeling and poorly defined molecular origins. However, the exact causal role of inflammation in PAH has been debated with the possibility that inflammation more clearly governs PAH severity rather than the overall risk.

[0081] Molecular homeostasis-promoting factors and brakes that regulate inflammation are important for maintaining cellular functions. However, specific levers that control inflammation, which causes EC dysfunction such as PAH, have not yet been fully described. Lysosomal activity has gradually come to be recognized as a major regulator of inflammation, and lysosomal dysfunction has been observed in PAH. The core of maintaining the function of lysosomal enzymes is the proper acidification of the lumen space via the vacuolar H + ATPase (V-ATPase) family, and the loss of this hydrolytic ability leads to a group of diseases known as lysosomal storage disorders (LSDs) in which pulmonary vascular phenotypes have been reported. Nuclear receptor coactivator 7 (NCOA7) directly binds to and regulates V-ATPase activity, controls endolysosomal function, and has been reported to have functions in controlling the entry of bacterial and viral pathogens, the acidification of renal tubules, and the function of neurons. NCOA7 is upregulated in human ECs and PAH lung tissues by inflammatory stimuli, but the causal relationship linking NCOA7 to cardio-pulmonary vascular diseases has not been clarified.

[0082] Downstream of acidification, lysosomes possess pH-sensitive hydrolytic enzymes responsible for the degradation of cellular waste and the transport of macromolecules. The degradation of cellular waste via lysosomes is associated with autophagy, and this process may be related to PAH. In particular, the loss of lysosomal hydrolase activity leads to the accumulation of oxysterols and bile acids, which are bioactive molecules upregulated in the plasma and lungs of PAH patients. Oxysterols and bile acids affect cholesterol biosynthesis and cell membrane properties and promote important cellular defenses in adaptive and innate immunity. At the endothelial level, these molecules have the ability to activate endothelial immunity and contribute to peripheral vascular diseases such as atherosclerosis and hypertension. Also recently, in an unbiased plasma metabolome analysis of 2,796 PAH patients, a metabolome-wide association between signatures of glucuronidated oxysterols and downstream bile acids and both measures of PAH severity and mortality (P < 1.1×10 after correction) -6) has been revealed. Therefore, since NCOA7 is essentially related to both lysosomal biology and the metabolome-wide association between lysosome-derived oxysterols and bile acid levels and PAH mortality, the inventors sought to clarify whether NCOA7 controls oxysterol and bile acid metabolism, the inflammatory lung EC disease type, and the development of PAH. Elucidating these associations will provide a mechanistic explanation underlying the association of glucuronidated oxysterols and bile acids with PAH mortality and severity.

[0083] Method RNA extraction and quantitative polymerase chain reaction: Cells were lysed with QIAzol Lysis Reagent (Qiagen; 79306), and RNA was extracted using the Rnaeasy Kit (Qiagen; 74004). Complementary DNA was synthesized using the High-Capacity cDNA Reverse Transcription KIt (ThermoFisher; 4368813). Quantitative real-time PCR (RT-qPCR) was performed on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR instrument. The expression of the target gene was normalized to a housekeeping gene (i.e., ACTB), and the fold change was calculated using the 2 -ΔΔCt method. TaqMan™ Universal PCR Master Mix (ThermoFisher; 4305719) was used together with TaqMan primers (Table 1). PowerUp™ SYBR™ Green Master Mix (ThermoFisher; A25742) was used together with custom-designed primers (Table 2).

[0084] [Table 1]

[0085] [Table 2]

[0086] Immunofluorescence staining of lung tissue: Lung tissue embedded in OCT was sliced at a thickness of 5 - 7 microns using a cryostat and then mounted on gelatin-coated histological slides. The sections were rehydrated with PBS for 5 minutes, fixed with 2% PFA for 30 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 5% donkey serum and 2% BSA in PBS at room temperature for 1 hour. The primary antibody (Table 3) was diluted with 2% BSA and incubated overnight at 4°C. The AlexaFluor-conjugated secondary antibody was used at a dilution rate of 1:1000 in 2% BSA at room temperature for 1 hour (ThermoFisher). Next, the sections were counterstained with Hoescht at room temperature for 1 minute and then mounted. Small pulmonary blood vessels not related to the bronchial airway (diameter 30 - 100 microns) were selected for imaging.

[0087]

Table 3

[0088] Single-cell transcriptomics: Single-cell RNA sequencing was performed on the lungs of healthy controls and patients with idiopathic PAH. The expression matrix was derived using CellRanger. Subsequent batch correction, scaling, and normalization were all performed using SCTransform of Seurat v3. Cell types were determined using SingleR with the Blueprint ENCODE reference. When the transformed expression value was greater than 0, the cells were identified as clearly expressing NCOA7. When the transformed expression value was greater than 0.2, the cells were identified as NCOA7-expressing cells.

[0089] Generation of iPSCs and CRISPR-Cas9 gene editing: To generate isogenic lines, the G allele of SNP rs11154337 was introduced into control iPS cells using pSpCas9(BB)-2A-GFP (PX458; Addgene; 48138) as a genome editing vector. For site-specific CRISPR-Cas9 and guide RNA sequence construction, two reverse complementary guide oligos were annealed and ligated into the linearized PX458 vector (Table 4). Single-stranded oligodeoxynucleotide (ssODN) templates with mutation / repair sites were designed as previously described (F. A. Ran et al., Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308 (2013)). Control iPSCs were dispersed as single cells at a confluence density of 40-50% the day before transfection. The next day, CRISPR-Cas9 and the ssODN template were transfected into iPSCs using GeneJammer reagent according to the manufacturer's protocol (Agilent Technologies 204132). After 36 hours, GFP-positive cells were sorted using FACS and seeded into 6-well plates at a density of 2,000 cells / well. After 1 week of growth, single-cell-derived colonies were observed and sorted for genotyping characterization and subsequent growth (Table S4). DNA was extracted from the harvested cells using QuickExtract solution (Epicenter). PCR amplification was performed using PrimeSTAR® GXL DNA Polymerase (Clontech) to determine the genotype of the edited region.

[0090]

Table 4

[0091] Differentiation of iPSCs into endothelial cells: Generation of iPSC-ECs is carried out using a chemical differentiation protocol, which consists of three main steps: mesoderm induction, endothelial specification, and iPSC-EC purification. Briefly, mesoderm induction was performed by activating Wnt signaling using the glycogen synthase kinase-3β inhibitor CHIR99021 (Selleckchem; S2924) in RPMI medium (Life Technologies; 11875-093) without insulin supplementation from B-27 (Life Technologies; A18956-01). Insulin is thought to have an adverse effect on mesoderm induction, so the use of B27 without insulin is considered to improve differentiation efficiency. Next, endothelial specification was performed by adding growth factors such as vascular endothelial growth factor (VEGF; 50 ng / mL; Gemini; 300196P) and fibroblast growth factor (FGF; 25 ng / mL; Gemini; 300113P) to EGM(TM)-2 Endothelial Cell Growth Medium-2 BulletKit(TM). The transforming growth factor-β (TGFβ) inhibitor SB431542 (10 μM; Selleckchem; S1067) was also added to increase the yield because it promotes EC generation and inhibits smooth muscle cell differentiation from endothelial progenitor cells. Finally, purification of iPSC-ECs was performed using magnetic-activated cell sorting (MACS) against the mature EC surface marker vascular endothelial (VE)-cadherin (also known as CD144). Mature iPSC-ECs were labeled with magnetic CD144 MicroBeads (Miltenyi Biotech; 130-097-857), captured on a column in a magnetic field, and then separated from unlabeled cells. The purified iPSC-Ecs were maintained in EGM(TM)-2 Endothelial Cell Growth Medium-2 BulletKit(TM).

[0092] Characterization of iPSC-Ec by flow cytometry: CD144 +Cells before and after purification were analyzed using flow cytometry for endothelial surface markers. Specifically, the expression of the mature endothelial progenitor marker CD34 (FITC anti-CD34 clone 581; BD Pharmingen™; 555821), the mature endothelial marker VE-cadherin / CD144 (APC anti-CD144 clone Bv9; BioLegend; 348508), and vascular endothelial growth factor receptor 2 (VEGFR2, or CD309; PE anti-CD309 clone 89106; BD Pharmingen™; 560872) was evaluated (Table 3). Samples were measured on a BD LSRFortesssa™ Flow Cytometer (BD Biosciences) at the University of Pittsburgh's Unified Flow Core.

[0093] Characterization of iPSC-EC by immunofluorescence staining: CD144 + After purification of + , iPSC-EC were further characterized by immunofluorescence staining of cell surface markers. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Cells were stained overnight at 4°C with anti-VE-cadherin / CD144 antibody (1:100; abcam; ab33168) or anti-CD31 (also known as platelet and endothelial cell adhesion molecule-1; PECAM-1; 1:100; abcam; ab24590) (Table 3). After rinsing with PBS, cells were incubated with the appropriate secondary antibody (1:1000) in 5% BSA for 1 hour at room temperature. Cells were rinsed with PBS and mounted with ProLong™ Gold Antifade Mountant containing DAPI (ThermoFisher; P36935). Images were acquired with a Nikon A1 confocal microscope.

[0094] Characterization of iPSC-ECs by in vitro tube formation: To confirm the endothelial phenotype, a capillary-like tube formation assay was performed using an in vitro Angiogenesis assay (R&D Systems; 3470-096-K) (DeCicco-Skinner et al., 2014). Basement membrane extract with reduced growth factors was seeded onto 96-well plates and solidified at 37°C for 30 minutes. Subsequently, iPSC-ECs were seeded (20,000 cells / well) into wells containing cell-type specific medium without growth factors and serum. After 6 hours, capillary-like structures were imaged at 10x magnification using an EVOSTM XL Core Imaging System (ThermoFisher).

[0095] Transfection of cells for RNA silencing: Human PAECs were transfected at an approximate confluency of 70 - 80% with 20 nM of negative control (siNC) or target gene (siGene) silencing RNA (siRNA) (Table 5). Lipofectamine® 2000 (ThermoFisher; 11668019) was mixed with siRNA according to the manufacturer's protocol. The Lipofectamine®:siRNA mixture was incubated with human PAECs in Opti-MEMTM reduced serum medium (ThermoFisher; 31985062) for 4 - 6 hours. After incubation, the transfection medium was removed and replaced with cell-specific growth medium containing full serum. Experiments were performed 48 hours after transfection.

[0096]

Table 5

[0097] Construction of lentiviral plasmids and particles: cDNA sequences encoding full-length NCOA7 (mRNA transcript variant 1, NM_181782.5) and short-length NCOA7 (mRNA transcript 6, NM_001199622.2) were amplified by PCR using HindIII and NheI linkers. The PCR products were directly cloned downstream of the Myc-tagged green fluorescent protein (mGFP) open reading frame of the pmGFP-ADAR1-p110 vector (Addgene; 117928). The cDNA sequences encoding mGFP-fused full-length and short-length NCOA7 were further subcloned into the pCDH-CMV-MCS-EF1α-Puro lentiviral expression vector (Systems Biosciences; CD510B-1). The cloned plasmids were confirmed by DNA sequencing at the Unified Flow Core of the University of Pittsburgh.

[0098] HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. HEK293T cells were transfected using Lipofectamine® 2000 (ThermoFisher; 11668019) with a lentiviral plasmid containing the target gene (or an empty vector as a control virus) and packaging plasmids from the ViraPower™ Lentiviral Packaging Mix (ThermoFisher; K497500). Viral particles were harvested 48 hours after transfection, pelleted, and then filtered.

[0099] Transduction of cells for lentiviral vector delivery: Human PAECs were transduced by directly applying polybrene (8 μg / mL) containing an empty control vector expressing GFP or viral particles with the target gene to the medium. The transduction efficiency was evaluated by direct measurement of GFP expression and transcript and protein expression. Experiments were performed 72 hours after transduction.

[0100] Protein extraction and immunoblotting: Cells were rinsed twice with PBS and then harvested in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the PierceTM BCA Protein Assay Kit (ThermoFisher; 23225). Protein lysates were separated using 4–15% Mini-PROTEAN® TGXTM Precast Protein Gels (Bio-Rad Laboratories; 4561086) and then transferred to PVDF membranes. Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Next, primary antibodies were added and incubated overnight at 4 °C (Table 3). The following day, blots were washed three times with TBST for 10 min each. Next, blots were incubated with the appropriate secondary antibody conjugated to HRP for 1 h at room temperature. After another TBST wash, blots were visualized using Pierce ECL reagent and images were captured using a BioRad ChemiDoc XRS+.

[0101] Chromatin immunoprecipitation and quantitative polymerase chain reaction: The MAGnify® Chromatin Immunoprecipitation System (Invitrogen; 49-2024) was used according to the manufacturer's protocol. Briefly, 1 × 10 6 human PAECs or iPSC-derived ECs were used for each ChIP reaction. Dynabeads® were conjugated with rabbit IgG antibody (1 μg / μL) or rabbit NF-κB p65 antibody (5 μg / μL) for 1 h at 4 °C (Table 3). 1 × 10 6The cells were treated with trypsin, pelleted, and resuspended in 500 μL for each reaction. Each reaction was cross-linked with 1% methanol-free formaldehyde at room temperature for 10 minutes. The cross-linking reaction was inhibited with 1.25 M glycine at room temperature for 5 minutes. From this point on, all steps of the reaction were maintained at 4°C. The samples were pelleted and washed three times with cold PBS. Then, each reaction was resuspended in 50 μL lysis buffer containing protease inhibitors, followed by chromatin shearing. The cells were sheared using a protocol of repeating 6 cycles of 20 seconds on and 40 seconds off with a Biorupter® UCD-200. The samples were pelleted and the supernatant containing the chromatin products was collected and confirmed to be appropriately fragmented on a DNA gel. Antibody-conjugated Dynabeads® were incubated with the chromatin at 4°C for 2 hours with inversion rotation. Next, the samples were washed in an immunoprecipitation buffer system and then the cross-links were reversed with proteinase K. Next, the DNA was purified and then quantitative PCR was performed. The primers used for ChIP-qPCR are shown in Table 6.

[0102]

[0103] Chromatin conformation capture (3C) assay: The 3C assay was performed. Briefly, 1×10 7 ​Human PAECs were crosslinked with 1% formaldehyde for 10 minutes at room temperature. Nuclei were isolated and genomic DNA was digested overnight at 37 °C with 400 U of BspHI at 950 rpm. Prediction based on putative BspHI cleavage sites on the genome indicated that restriction enzyme digestion would generate a 1,666 bp genomic DNA fragment containing SNP rs11154337 and a 9,269 bp DNA fragment containing the promoter of NCOA7. The digested DNA was diluted and ligation was performed in a 16 °C water bath for 4 hours with or without 4000 U of T4 DNA ligase. Subsequently, the crosslinked DNA was reversed overnight at 65 °C with 100 μg of proteinase K. Next, the DNA was isolated and purified. PCR was performed using primers selected to target the potential ligation fusion sequences of the DNA fragment containing SNP rs11154337 and the fragment containing the promoter of NCOA7 and to be proximal to the BspHI site: (1) 50 bp from the BspHI site at the 3' end of the NCOA7 promoter fragment, 5’TTT GGG CAA TGT TAC AGC AA-3’ (forward primer (SEQ ID NO: 28)) and (2) 57 bp from the BspHI site at the 5' end of the SNP rs11154337 fragment, 5’-GAA ATG CCA GGG ATT CCT TA-3’ (reverse primer, (SEQ ID NO: 29)). The amplification product was a 107 bp fragment, confirming the presence of the fusion sequence. The PCR products were separated by gel electrophoresis and analyzed by DNA sequencing.

[0104] Transcriptome analysis of human PAEC and data acquisition: Microarray data were obtained using the Affymetrix Clariom D Human Array at the Genomics Research Core of the University of Pittsburgh. The microarray chips were performed on a total of 12 samples with 4 groups and 3 replicates. PAEC was performed for either the control group or the knockdown of the NCOA7 gene. Furthermore, thereafter, each group was either left under control conditions or further stimulated with the inflammatory cytokine IL-1β for 24 hours. The raw data were processed using the Bioconductor package in the R language, and a list of differentially expressed genes was created using Benjamini-Hochberg corrected p-values less than 0.05 to minimize the false discovery rate (FDR). The following add-on packages: oligo, limma, affycore tools, gplots, pd.clariom.d.human were utilized for the analysis of this microarray data. Very roughly speaking, the flow of this program is to convert the raw microarray data in.CEL format into a list of differentially expressed genes. The workflow included loading the raw data into R, filtering and normalizing the raw data, plotting to show the homogeneity between samples, adding annotations, setting up a comparison matrix to perform statistical analysis between groups, generating a list of differentially expressed genes, filtering the said list, constructing a heatmap to depict the generalized trends, and including the overlap between the generated gene list and the inventors' PH Network. Next, gene set enrichment analysis (GSEA) of the identified differentially expressed genes was performed using Gene Ontology, REACTOME, KEGG, and BioCarta.

[0105] Proximity ligation assay: The direct interaction between NCOA7 and the V-ATPase subunit ATP6V1B2 was evaluated using the Duolink® Proximity Ligation Assay (Millipore Sigma; DUO92102). Human PAECs were seeded in Nunc® Lab-Tek® II Chamber Slide® System (20,000 cells / well; ThermoFisher; 154453) and then fixed with 4% paraformaldehyde for 15 minutes. After permeabilization and blocking according to the manufacturer's protocol, the wells were incubated overnight at 4°C with mouse anti-NCOA7 antibody (Santa Cruz Biotechnology; sc-393427), rabbit anti-ATP6V1B2 antibody (abcam; ab73404), both antibodies, or no antibody (Table 3). Cells were then incubated with PLUS and MINUS probes for 1 hour, ligated for 30 minutes, and amplified at 37°C for 100 minutes. The slides were then mounted with ProLong® Gold Antifade Mountant containing DAPI (ThermoFisher; P36935). Images were acquired with a Nikon A1 confocal microscope at the Center for Biologic Imaging, University of Pittsburgh.

[0106] Transmission electron microscope: Human PAECs grown on tissue culture plasticware were fixed overnight at 4°C in 2.5% glutaraldehyde in 100 mM PBS (8 g / L NaCl, 0.2 g / L KCl, 1.15 g / L Na2HPO4·7H2O, 0.2 g / L KH2PO4, pH 7.4). After washing the monolayer three times with PBS, post-fixation was performed in 1% osmium tetroxide aqueous solution and 1% Fe6CN3 for 1 hour. After washing the cells three times with PBS, dehydration was carried out in a 30 - 100% ethanol series while changing Poly / Bed® 812 embedding resin (Polysciences) several times. The culture was embedded by inverting a BEEM® capsule filled with Poly / Bed® 812 over the cells. The block was cured overnight at 37°C and then cured for 2 days at 65°C. The monolayer was peeled off from the coverslip and re-embedded to obtain cross-sectional sections. Ultra-thin sections (60 nm) of the cells were obtained with a Riechert / Leica UltraCut E ultramicrotome and post-stained with 4% uranyl acetate for 10 minutes and then 1% lead citrate for 7 minutes. The sections were observed at 80 kV with a JEOL JEM-1400Flash transmission electron microscope. Images were taken with a bottom-mounted AMT digital camera. The obtained microscope images were manually analyzed under blind conditions. Lysosome area was quantified using Fiji.

[0107] Evaluation of lysosomal hydrolase activity: Lysosomal activity and function were evaluated using measurements of enzyme activity. Human PAECs were plated on glass coverslips and stained for total lysosome measurement. In the LysoLive assay (Marker Gene Technologies, Inc.; M27745), the β-glucosidase-specific substrate GlucGreen was incubated in the medium at 5 μM for 30 minutes at 37°C. After washing the cells three times with ice-cold PBS, they were fixed with 4% PFA for 15 minutes at room temperature. The slides were mounted with ProLong® Gold Antifade Mountant containing DAPI (ThermoFisher; P36935).

[0108] In the SiR-lysosome assay (Cytoskeleton, Inc.; CYSC012), a cell-permeable peptide conjugated to the silicone rhodamine (SiR) dye was incubated in human PAECs as an indicator of active cathepsin D. The cells were incubated with SiR-lysosome at 1 μM and the calcium channel blocker verapamil at 1 μM for 30 minutes at 37 °C to enhance the signal intensity. The cells were rinsed three times with ice-cold PBS, fixed with 4% PFA, and mounted as described above.

[0109] Evaluation of lysosome acidification: Lysosome acidification was measured using the LysoSensor™ Yellow / Blue DND-160 (PDMPO) dye (ThermoFisher; L7545). The LysoSensor™ Yellow / Blue DND-160 (PDMPO) dye exhibits pH-dependent (pK a 4.2) dual excitation (i.e., 329 and 384 nm) and dual emission (i.e., 440 and 540 nm) spectral peaks. In acidic organelles, this dye has mainly yellow fluorescence. In basic organelles, the dye has mainly blue fluorescence. The unique spectral properties of this dye enable ratiometric quantification.

[0110] Human primary PAECs were incubated with 1 μM of the dye in 0.1% FBS cell-specific medium at 37 °C for 1 hour. The cells were rinsed with PBS, treated with trypsin, pelleted at 300 g for 5 minutes in a polystyrene tube, and rinsed two more times with PBS. The cells were immediately analyzed on a BD LSRFortesssa™ flow cytometer (BD Biosciences) at the University of Pittsburgh's Unified Flow Core. The median fluorescence intensity (MFI) ratio was calculated using the yellow MFI fluorescence value relative to the blue MFI fluorescence value.

[0111] Evaluation of lysosomal lipid content: Neutral lipids were stained with the fluorescent dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®; ThermoFisher; D3922). Acidic organelles (i.e., lysosomes) were stained with LysoTracker® Red DND-99 (ThermoFisher; L7528). Human primary PAECs were incubated at 37 °C for 30 minutes in cell type-specific medium containing 1 μM BODIPY® and 50 nM LysoTracker® Red DND-99. Cells were then washed three times with PBS and fixed with 4% PFA for 30 minutes at room temperature. After further washing the cells three times with PBS, they were mounted with ProLong® Gold Antifade Mountant containing DAPI (ThermoFisher; P36935). Images were acquired with a Nikon A1 confocal microscope at the Center for Biologic Imaging, University of Pittsburgh.

[0112] The lysosomal lipid content was measured by the degree of co-localization of BODIPY® (i.e., neutral lipids) and LysoTracker® Red DND-99 (i.e., acidic organelles). Co-localization was measured using EzColocalization in Fiji and quantified as Pearson's correlation coefficient.

[0113] Targeted LC-MS of cholesterol intermediates and oxysterols: Human PAECs were treated and, for cholesterol intermediate and oxysterol analysis, per 16 x 125 mm glass tube (Pyrex; 9826), 1 x 10 6Cells were collected. Liquid-liquid extraction of sterols was performed on the cells. Briefly, 1 mL of dichloromethane, methanol, and water were added to each sample. The sample was then vortexed and centrifuged to obtain two liquid phases. The lower phase was carefully transferred to a new glass tube and dried under nitrogen. Next, the sample was resuspended in hexane, and the resulting lipid species were analyzed by liquid chromatography-mass spectrometry (LC-MS). The samples were analyzed on a SCIEX QTRAP 6500+ equipped with a Shimadzu LC-30AD HPLC system and a 150×2.1 mm, 5 μm Supelco Ascentis silica column. LC-MS data were analyzed using MultiQuant (SCIEX).

[0114] Statistical analysis of the UPMC cohort and the STRIDE cohort: For the UPMC cohort, the effect of the SNP rs11154337 G minor allele on the 6-minute walk distance was calculated using the dominant genetic model. Furthermore, the effect of this minor allele on the time to death or final follow-up was tested using the Cox proportional hazards model. In both models, the genetic effects were adjusted for sex, age, comorbidities, and vasodilator therapy. Stata 17.0 pymol was used for these analyses.

[0115] In the STRIDE cohort, the analysis was started from the raw data in plink format. The strand information was converted to InfiniumOmniExpress-24v1-3_A1-b37.strand using update_build.sh. Next, phasing was performed using shapeit, and impute2 was executed using 1000G_Phase3 b37 for imputation. Chromosome bins were merged, and redundant SNPs were removed using gtool. The first pass of the survival analysis was performed using the R package gwasurvivr, which conducts a coxph test for each SNP against the time to death (FinalEvent), with covariates: gender, age, PAH type, WHO classification, study inclusion (Encysive or Prospective), AnyDrugsBefore, UsePDE, UsePros, UseWarf, UseOxy, and a maf filter of 0.005. Additionally, SNP rs11154337 was tested against the time to death (FinalEvent) in European patients (EthConEUR) based on self-reported ethnicity, and discriminant principal component analysis was performed.

[0116] Staining of neutral lipids: Intracellular neutral lipids were stained using the fluorescent dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®). Live cells were cultured on plastic products with a glass coverslip placed on top. The cells were washed three times with PBS to remove residual culture medium. A staining solution of 2 μM BODIPY® in PBS was applied to the cells at 37 °C for 15 minutes. After rinsing the cells three times with PBS, they were fixed with 4% paraformaldehyde at room temperature for 15 minutes. The coverslip was mounted with ProLong® Gold Antifade Mountant containing DAPI (ThermoFisher; P36935). Images were acquired using a Nikon A1 confocal microscope at the Center for Biologic Imaging, University of Pittsburgh.

[0117] Measurement of cholesterol uptake: To evaluate cholesterol uptake, a cholesterol uptake assay kit was utilized according to the manufacturer's specifications (abcam; ab2362). Briefly, treated human PAECs were incubated for 24 hours in cell type-specific medium supplemented with 0.1% serum and 20 μg / mL of fluorescent NBD-cholesterol. Cells were rinsed with PBS, treated with trypsin, pelleted at 300 g for 5 minutes in a polystyrene tube, and rinsed two more times with PBS. Cells were immediately analyzed on a BD LSRFortesssa™ Flow Cytometer (BD Biosciences) at the University of Pittsburgh's Unified Flow Core. Flow cytometry analysis was chosen over confocal microscopy due to the high photobleaching rate observed with NBD-cholesterol.

[0118] Evaluation of cholesterol content: To evaluate cholesterol content, the Cholesterol / Cholesterol Ester-Glo™ assay kit (Promega; J3190) was utilized. In this assay, cholesterol is measured using cholesterol dehydrogenase, which couples the presence of cholesterol to the production of NADH and, in turn, the activation of pro-luciferin. Human PAECs were seeded at a density of 20,000 cells / well in a 96-well plate in six replicates. The assay was performed as specified by the manufacturer.

[0119] Apoptosis was measured by caspase-3 / 7 activity: Apoptosis was evaluated using the Caspase-Glo® 3 / 7 assay system (Promega; G8090). This assay functions by providing a luminescent caspase-3 / 7 substrate optimized for caspase activity. When this substrate is cleaved, a luminescence-based signal is generated via luciferase. An equal volume of this substrate was added to wells containing human PAECs (5,000 cells / well) and incubated at room temperature for 30 minutes. Luminescence was measured using a spectrophotometer. The luminescence signal was normalized by the protein content per well evaluated using the Pierce™ BCA Protein Assay Kit (ThermoFisher; 23227).

[0120] Proliferation was measured by BrdU incorporation: Proliferation was evaluated using the BrdU Cell Proliferation assay kit (Cell Signaling Technology; 6813). This assay functions by measuring 5-bromo-2'-deoxyuridine (BrdU) in proliferating cells using an anti-BrdU antibody. BrdU was added to complete growth medium containing 5% serum for 2 hours. Human PAECs (5,000 cells / well) were fixed and denatured, and then a mouse anti-BrdU antibody was applied. Next, an anti-mouse HRP-conjugated antibody was added. Next, a chromogenic substrate was added to detect the antibody complex with BrdU to which HRP was bound. Absorbance was measured at 450 nm using a spectrophotometer.

[0121] Leukocyte and monocyte adhesion assay: Endothelial immune activation was evaluated by measuring the adhesion of immune cells to the endothelial monolayer. Human PAECs were cultured until a complete monolayer was formed. Immune cells were stained with CellTrace™ Blue or CFSE (ThermoFisher; blue, C34568; CFSE, C34554) according to the manufacturer's protocol. 2.0 - 2.5×10 5Individual stained immune cells were added to each well of a 6-well plate and incubated for 24 hours. Subsequently, the wells were rinsed twice with PBS and then fixed with 4% PFA at room temperature for 15 minutes. After fixation, the cells were rinsed once more with PBS. Fluorescent images of each well were acquired at a magnification of 4x. The number of immune cells per image was quantified using Fiji. For the leukocyte adhesion assay, HuT 78 cutaneous T lymphocytes (ATCC) were used. For the monocyte adhesion assay, THP-1 peripheral blood monocytes (ATCC) were used.

[0122] Application of oxysterols and bile acids: Oxysterols and bile acids were applied to human PAECs in 0.1% FBS, cell-specific medium. 25-Hydroxycholesterol and 7HOCA were dissolved in 100% ethanol and applied to the cells at concentrations of 25 μM or 50 μM for 24 hours.

[0123] Animal studies: All animal studies were approved by the Division of Laboratory Animal Resources at the University of Pittsburgh. The Ncoa7 knockout mouse strain (C57BL / 6 Ncoa7tm1.1(KOMP)Vlcg) was obtained from the Knockout Mouse Project (KOMP; komp.org) and generated at the Genome Editing, Transgenic, & Virus Core of the Magee Women’s Research Institute using sperm for reactivation. The resulting mice were bred in-house to generate homozygous Ncoa7 knockout mice. To induce a severe PH-producing lung inflammation model, Ncoa7 knockout mice were mated with C56BL / 6 Il6 transgenic (Tg + ) mice. Il6 Tg +Mice have the Clara cell 10kD promoter (CC10) that promotes constitutive expression of IL-6 in the lungs (M. K. Steiner et al., Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104, 236-244, 228p following 244 (2009)). C57BL / 6 mice were used for the oral intermittent delivery of PBS or 7HOCA (10 mg / kg), and were continuously injected every 5 days for 4 weeks under chronic hypoxia (10% O2). After the mice were allowed to progress to 15 weeks of age under normal oxygen concentration, echocardiography, invasive hemodynamic measurements, and tissue collection were performed. Using the monocrotaline rat model of PAH, a single injection of monocrotaline (80 mg / kg) was given at 8-9 weeks of age. Thereafter, recordings were taken after intraperitoneal injection of DMSO or 958 ami (7.5 mg / kg) 10 days after monocrotaline challenge in the rats.

[0124] Measurement of hemodynamics: Echocardiography was performed on 15-week-old mice using a 15-45 MHz transthoracic transducer and a VisualSonics Vevo770 system (Fujifilm). Anesthesia was administered with 2% isoflurane in 100% O2 during animal positioning and hair removal, and then reduced to 0.8% isoflurane during image acquisition. Data analysis was performed by a technician under blinded conditions.

[0125] For right heart catheterization, mice were administered ketamine / xylazine (9:1; Henry Schein) or isoflurane (Henry Schein). The isoflurane vaporizer was maintained at 1.5-2% with an oxygen gas flow rate of 1 L / min. Right ventricular systolic pressure was measured using a Millar catheter (SPR-513 and SPR-671). The catheter was inserted into the jugular vein and then guided from the right atrium into the right ventricle. A stable right ventricular systolic pressure waveform was measured for 2 minutes. Waveform analysis was performed under blinded conditions.

[0126] NCOA7 short Structural modeling of the catalytic domain of: NCOA7 short(Isoform 5; Q8NI08-5)'s sequence was downloaded from UniProt. Its catalytic domain (P55-D219) was modeled using SWISS-MODEL based on the crystal structure (PDB ID 4ACJ) of the TLDc domain of the oxidation-resistant protein 2 (OXR2) from zebrafish. The sequence identity between the catalytic domains of NCOA7 and OXR2 was calculated to be 61.8% using Clustal Omega, which means that the two domains share the same structure.

[0127] Gaussian network model (GNM) analysis: In GNM analysis, the protein structure is represented as an elastic network, and residues function as nodes whose positions are specified by the positions of α-carbons. Thus, the GNM of NCOA7 was developed using a total of 165 residues. The overall potential was expressed as the sum of harmonic potentials between pairs of nodes within an interaction range defined as a C α -C α distance less than 7.3 Å. The topology of the resulting network was recorded with an N×N Kirchhoff matrix. All calculations were performed using the ProDy API.

[0128] Drug-likeness simulation and analysis: Using the all-atom MD simulation package NAMD, drug-likeness simulations of NCOA7 in the presence of probe molecules were performed using the CHARMM36 force field for the protein, the TIP3P water model, and the CGenFF force field for the probe molecules. The probe molecules were benzene, isobutane, imidazole, acetamide, isopropanol, isopropylamine, and acetic acid, obtained from a statistical evaluation of the most frequently seen chemistries / functional groups in FDA-approved drugs. Trajectories were analyzed using ProDy's DruGUI module, and six independent 40-ns runs were performed. All MD snapshots were C αThe atoms were superimposed on the reference PDB structure, and a spatial representation based on a cubic lattice with a grid edge size of 0.5 Å was used. Probe molecules with non-hydrogen atoms within 4.0 Å of the protein atoms were considered to interact with the protein. For each probe type, the individual occupancies of the lattice were calculated using its centroid. The occupancy of each probe for each voxel was evaluated, and the binding energy was quantified using the inverse Boltzmann relationship. From the obtained binding free energy map, interaction spots with low (favorable) energy for one or more probe types were identified, and voxels with high occupancy were called druggable hotspots.

[0129] Pharmacophore modeling: Using Pharmmaker, the residues involved in high-affinity interactions with each molecular probe type were identified. Residue-probe interactions were then ranked based on their frequency of occurrence at druggable hotspots in multiple runs. Snapshots showing potential residue-probe pairs simultaneously (e.g., N62-benzene, E66-isopropylamine, P80-benzene, and W81-benzene) were selected as templates for constructing the pharmacophore model. This pharmacophore model included a hydrogen bond donor and hydrophobic features at the isopropylamine site, as well as hydrophobic and aromatic rings at two benzene sites. Next, this pharmacophore model was screened against the ZINC and MolPort libraries using Pharmit. The MolPort library contains 67,033,884 conformers corresponding to 4,848,718 compounds, and the ZINC library contains 122,276,899 conformers of 13,127,550 compounds. Among the top-scoring compounds, MolPort-004-267-958 was selected for further refinement after the first experimental validation.

[0130] 958 and 958 amiMolecular Dynamics Simulations: All-atom MD systems were set up using the GHARMM-GUI Solution Builder. Simulations were performed using the MD simulation package NAMD, with the CHARMM36m force field for proteins, the TIP3P water model, and the CGenFF force field for compounds. For 958 and 958 ami , three independent runs of 0.2 ms each (total 0.6 ms) were performed. The systems in each case were composed of the NCOA7 protein and the compound in the presence of explicit water and 0.15 M NaCl, and were relaxed using the CHARMM-GUI equilibration steps. NPT dynamics was performed for 0.2 ms at a time step of 2 fs, constant pressure (1 bar), and temperature (300 K). Contact times between the target and the compound and hydrogen bond formation were analyzed using VMD 1.9.4. Data visualization was performed using PyMOL 2.3.5 and GNUPlot. Binding affinity was calculated using PRODIGY-LIG.

[0131] Statistics: All in vitro data represent at least three independent experiments. The number of animals used in a given experimental model was calculated to detect an 80% power and a 10% standard deviation, measuring at least a 20% difference between the mean values of the control and experimental groups. The number of patient samples used for molecular analysis was mainly determined by clinical availability. The Shapiro-Wilk test was used to determine the normality of the data distribution. For normally distributed data, paired data were analyzed using Student's two-sided t-test, and grouped data were compared using one-way or two-way ANOVA with Tukey's post hoc analysis to correct for multiple comparisons. Significance was determined by a P-value of less than 0.05. All data are presented as mean ± standard deviation.

[0132] Results Convergent Inflammatory Regulation of NCOA7 in PAH Cell, Animal, and Human Cases: Unbiased transcriptome analysis was performed on primary human pulmonary artery endothelial cells (PAECs) exposed to interleukin-1β (IL-1β), an inflammatory cytokine that is elevated in the plasma of PAH patients and is known as an inducer of disease etiology. Lysosomal regulatory genes were globally upregulated, along with a distinct subset containing the V-ATPase subunit, which is a binding partner of NCOA7 that promotes lysosomal acidification (Figure 4(A)). Correspondingly, IL-1β upregulated both NCOA7, namely the canonical full-length isoform (NCOA7 full ), and to a greater extent, the short-chain length isoform of alternative initiation NCOA7 (NCOA7 short ) (Figure 4(B, C), Figure 5A(A)).

[0133] Other inducers of EC dysfunction in PAH also upregulated NCOA7 similarly. Specifically, exposure to the inflammatory cytokine IL6 and its soluble receptor (IL6Rα) associated with PAH induced both short-chain and full-length isoforms (Figure 5A(B, C)). Hypoxia, a well-established promoter of PH, also increased both isoforms (Figure 5A(D, E)). Collectively, these data indicate a potential role of NCOA7, particularly its unique alternative initiation isoform, across multiple inducers of PH.

[0134] Using a severe inflammatory rodent model of PAH, transgenic mice under constitutive IL-6 overexpression and chronic hypoxia showed increased Ncoa7 expression in CD31 + ECs isolated from lung tissue (Figure 5A(F, G)). This was also seen in the pulmonary vasculature of IL6 transgenic mice without hypoxia - a mild form of experimental PAH (Figure 4(D, E)). Similarly, examining the in situ localization of NCOA7 revealed prominent and transmural upregulation in the pulmonary vasculature, along with enhanced endothelial localization expression, in both the chronic hypoxia mouse model and the monocrotaline-exposed PAH rat model (Figure 5A(H - K)).

[0135] Furthermore, in the human lung tissues of Group I PAH patients, increased NCOA7 expression was observed in the pulmonary vessels compared with healthy controls (Figure 4 (F, G)). Single-cell RNA sequencing performed on the lungs of idiopathic Group I PAH patients showed an increase in the number of NCOA7-expressing ECs compared with healthy controls (22.58% vs. 29.66%) (Figure 4 (H)). Furthermore, in NCOA7-positive ECs, NCOA7 expression was upregulated in PAH patients (Figure 4 (I)). Thus, in primary PAECs, rodent models, and inflammatory models of PH using human patients, NCOA7 was found to be upregulated in the pulmonary vessels and, most importantly, in the endothelium. However, since loss of NCOA7 results in loss of lysosomal acidification and, in other contexts, increased inflammation and disease exacerbation, NCOA7 is thought to act as a homeostatic brake under inflammatory stress, suppressing disease pathogenesis by attenuating EC immune activation.

[0136] To investigate the upstream inflammatory mechanisms regulating NCOA7, binding sites for the well-established inflammatory transcription factor complex, namely, the RelA / p65 (RELA) subunit of NF-κB, were predicted within the canonical (i.e., full-length) and non-canonical (i.e., short isoform) NCOA7 promoter regions. Correspondingly, knockdown of RELA in PAECs suppressed the upregulation of both isoforms via IL-1β (Figure 4 (J, K)). A combination of chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) revealed significant enrichment of RelA / p65 in the DNA sequences of the canonical and non-canonical promoter regions (Figure 4 (L)), suggesting direct binding of the promoter and the transcription factor.

[0137] Deficiency of NCOA7 promotes lysosomal dysfunction and lipid accumulation: To investigate the putative relationship between NCOA7 and oxysterol production in the presence of inflammatory pathologies, we characterized the control of lysosomal acidification via NCOA7, considering the known function of lysosomes in sterol transport. In human PAECs exposed to IL-1β, knockdown of NCOA7 revealed interleukin-specific changes in the network of genes governing lysosomal function (Figure 6A(A)). In particular, many of these genes (e.g., ATP6V0A1, ATP6V1B2, ATP6V1C1, ATP6V1D, ATP6V1E1, ATP6V1G1, and ATP6V1H) encode subunits of the V-ATPase, a mechanism necessary for lysosomal acidification and thus the function of pH-sensitive enzymes.

[0138] Proteomics-based studies have revealed that NCOA7 interacts with ATP6V1B1, a kidney-specific paralog of ATP6V1B2. Correspondingly, in PAECs, knockdown of NCOA7 suppressed the IL-1β-mediated upregulation of ATP6V1B2, and forced overexpression of either the short or full-length isoform upregulated ATP6V1B2 (Figure 6A(B,C)). Furthermore, ATP6V1B2 was upregulated in the lung endothelium of PH rodent and human models (Figure 7A and 7B(A-H)). To assess the direct interaction between these proteins, proximity ligation assay showed perinuclear staining indicating the ATP6V1B2-NCOA7 interaction, which was consistent with the perinuclear distribution of lysosomes. Furthermore, short or NCOA7 full forced overexpression increased the number of ATP6V1B2-NCOA7 interactions in lysosomes (Figure 6A(D,E)). These data demonstrated the role of short and full-length NCOA7 as regulatory components of the V-ATPase complex, which is presumed to have downstream lysosomal function.

[0139] To evaluate the activity of NCOA7 in regulating lysosomal acidification, two quantitative measurements of lysosomal enzyme activity were used. First, the cleavage of the fluorescent LysoLive tracer was increased by IL-1β and then blocked by the deficiency of NCOA7 (Figure 6B (F, G)). Second, lysosome-dependent cathepsin D activity was evaluated using SiR-lysosome. Similar to the increase in ATP6V1B2 via IL-1β, cathepsin D activity was significantly upregulated by IL-1β as shown by SiR-lysosome fluorescence and was suppressed by knockdown of NCOA7 (Figure 6B (H, I)).

[0140] To directly evaluate lysosomal acidification, the acidotropic probe LysoSensor Green DND-189, which accumulates in acidic compartments and enhances fluorescence under acidic conditions, was utilized. Consistent with the increase in lysosomal enzyme activity via IL-1β, IL-1β increased the LysoSensor fluorescence signal, which was reversed by NCOA7 knockdown (Figure 6B (J)). Furthermore, IL-1β changed the fluorescence to yellow in PAECs when using the acidification probe LysoSensor Yellow / Blue DND-160, indicating promotion of lysosomal lumen acidification (Figure 6B (K)). The addition of NCOA7 deficiency reversed the yellow fluorescence shift via IL-1β. These findings are similar to those in LSDs that are noted for the accumulation of undigested cellular components in the lysosomal compartment.

[0141] Such failures in V-ATPase complex formation or lysosome acidification are known to be drivers of lysosomal morphological abnormalities. Thus, morphological analysis of lysosomes using transmission electron microscopy of NCOA7-deficient human PAECs revealed a marked hypertrophy of lysosomes quantified by lysosome area (Figure 6B (L, M)), indicating that the lysosomal compartment is unable to degrade and process cellular components. Furthermore, the enlarged lysosomes in NCOA7-deficient cells had lamellar inclusions indicative of lipid accumulation (Figure 6B (L, M); yellow arrows), again mimicking the LSD phenotype of abnormal lysosomal lipid accumulation.

[0142] To examine whether the lamellar structures seen in electron micrographs were lipids, PAECs were co-stained with a dye for neutral lipids (i.e., BODIPY (registered trademark)) and a dye specifically localized to acidic compartments (i.e., LysoTracker). In the absence of NCOA7, lipid spots increased throughout the cell and accumulation of vesicles was confirmed. Correspondingly, high concentrations of lipid spots were identified within acidic vesicles, supporting the idea that NCOA7 deficiency caused such lipid accumulation in lysosomes (Figure 6C (N, O)). Taken together, these findings confirmed that NCOA7 is a binding partner for the V-ATPase complex that promotes lysosome acidification and sterol transport in PAECs.

[0143] Deficiency of NCOA7 reprograms sterol metabolism via abnormal lipid accumulation: Changes in lysosomal lipid transport affect sterol homeostasis. Correspondingly, transcriptome analysis of NCOA7-deficient human PAECs revealed significant enrichment and downregulation of biosynthetic processes related to sterol metabolism (Figure 8A (A, B); red arrows). In sterol-saturated cells, the extracellular cholesterol uptake pathway is inhibited, particularly through a decrease in the density of low-density lipoprotein receptors (LDLRs) on the cell membrane. Deficiency of NCOA7 in PAECs decreases the expression of LDLR (Figure 8A (C)) and is accompanied by a functional attenuation of fluorescent-labeled cholesterol uptake (Figure 8A (D, E)). The total cholesterol content of NCOA7-deficient PAECs was also upregulated, but forced overexpression of NCOA7 decreased the total cholesterol content (Figure 8A (F, G)). No significant differences were detected in post-squalene intermediates between NCOA7-deficient PAECs and NCOA7-full-expression PAECs (Figure 9A and 9B (A~N)), indicating that the regulation of NCOA7-dependent sterol intermediate flux does not depend on de novo cholesterol synthesis. Therefore, the downregulation of sterol metabolism caused by NCOA7 deficiency is mainly promoted by lysosomal changes in sterol processing, rather than de novo synthesis.

[0144] To protect against cholesterol accumulation, cells can engage in its direct export via transporters or an increase in cholesterol solubility through a series of oxidation steps. Therefore, deficiency of NCOA7 significantly upregulated cholesterol 25-hydroxylase (CH25H), an oxysterol-generating enzyme that increases cholesterol solubility (Figure 8A (H)). The relevance of these processes in vivo was revealed, and CH25H was upregulated in the pulmonary vasculature of an inflammatory rodent model of PAH and Group I PAH patients and was associated with endothelial localization (Figure 8B (I~N) and Figure 9B (O, P)). Overall, these data established a central role for NCOA7 in maintaining EC sterol homeostasis through the production of oxidized sterol species in cultured PAECs and in vivo diseased endothelium.

[0145] NCOA7 deficiency induces endothelial production of oxysterols and downstream bile acid derivatives: To determine whether the observed upregulation of CH25H promoted oxysterol production, targeted lipidomic analysis was performed using liquid chromatography–mass spectrometry (LC-MS). Knockdown of NCOA7 in human PAECs exposed to IL-1β significantly upregulated 25-hydroxycholesterol (25HC), 27-hydroxycholesterol (27HC), and 7α-hydroxycholesterol generated by auto-oxidation (Figure 8B (O–Q)). These oxysterols are known to be metabolized to downstream bile acid derivatives by mechanisms that are not fully understood. Thus, knockdown of NCOA7 upregulated several downstream bile acid derivatives in the sequential pathway, such as 5-cholestene-3β-7α,25-triol, 5β-cholestane-3α,7α,12α-triol, and 5β-cholestane-3α,7α,12α,25,26-pentol (Figure 8B (R–T)). Furthermore, the upstream metabolites 3β,7α-dihydroxy-5-cholestenate and 7α-hydroxy-3-oxo-4-cholestenic acid (7HOCA) were also upregulated (Figure 8B (V, W)). Thus, consistent with the upregulation of the oxysterol-generating enzyme CH25H (Figure 8A (H)), NCOA7 deficiency induced the production of numerous oxidized cholesterol metabolites and downstream bile acids in ECs.

[0146] Oxysterols and bile acids as markers of morbidity and mortality in PAH: Given the emphasis on the clinical importance of this mechanism in the control of disease severity, a plasma signature containing NCOA7-dependent sterols and bile acids associated with PAH mortality was identified (adjusted P < 1.1 × 10 -6)。As a related study, unbiased metabolome-wide association studies from the multi-site PAH Biobank cohort (N = 2,796) identified 13 different plasma oxysterols and bile acids that best predicted 4-year mortality in PAH. In particular, among these top 13 oxysterols and bile acids, 4 were the same metabolites that were upregulated in NCOA7-deficient ECs (Figure 8B (Q, R, S, W)) and serum of Ncoa7-deficient mice (Figure 13(I)). These findings highlight the clinical importance of NCOA7-dependent oxysterols and bile acids in the control of PAH severity, thereby establishing a framework that links lysosome-dependent inflammation to population-level and metabolome-wide signals in PAH.

[0147] NCOA7 deficiency promotes endothelial dysfunction via oxysterol production: Considering the immunomodulatory function of oxysterols in diseased endothelium, we sought to determine whether NCOA7 deficiency promotes EC dysfunction in an oxysterol-dependent manner. NCOA7 deficiency upregulated vascular cell adhesion molecule 1 (VCAM1), a surrogate of endothelial immune activation (Figure 10A (A, B)). Conversely, forced overexpression of the NCOA7 isoform reversed VCAM1 expression (Figure 10A (C, E)). To determine whether NCOA7 deficiency depends on downstream oxidized cholesterol to induce the pathology of PAECs, we simultaneously performed knockdown experiments on the oxysterol-producing enzyme CH25H, whose expression was upregulated in NCOA7 deficiency. In particular, inhibition of CH25H induction under NCOA7 deficiency suppressed VCAM1 expression (Figure 10A (E, F)).

[0148] Similar to the changes in VCAM1 observed under various NCOA7 and CH25H expression states, increased adhesion of both leukocytes and monocytes to NCOA7-deficient EC monolayers was seen (Figure 10B (G, J)), which could be reversed by overexpression of NCOA7 (Figure 10B (H, K)), and adhesion of immune cells was significantly attenuated by inhibition of CH25H under conditions of NCOA7 deficiency (Figure 10B (I, L)).

[0149] Furthermore, consistent with the concepts of immune activation, apoptosis resistance, and hyperproliferative endothelium in pulmonary vascular disease, NCOA7 deficiency in PAECs suppressed apoptosis via IL-1β and simultaneously enhanced proliferative capacity (Figs. 11A(A, D)). NCOA7 promoted PAEC apoptosis under inflammatory conditions (Fig. 11A(B)), and in parallel, attenuated proliferation with more prominent inhibition under IL-1β (Fig. 11A(E)). However, inhibition of CH25H upregulation reversed the attenuation of apoptosis and enhanced the proliferative capacity of the cells (Figs. 11A(C, F)). Collectively, the presence of NCOA7 suppressed endothelial immune activation with induction of apoptosis and inhibition of proliferation.

[0150] To determine whether bile acids are sufficient to immunologically activate endothelium, 7HOCA was directly applied to PAECs in culture. Demonstrating its proinflammatory nature, 7HOCA significantly upregulated VCAM1 (Figs. 10C(M, N)), and consequently promoted the adhesion of both leukocytes and monocytes to the PAEC monolayer (Figs. 10C(O, P)). Notably, direct application of 25HC as well as downstream derivatives such as triols and tetrols similarly upregulated VCAM1 and promoted immune cell adhesion to the PAEC monolayer (Figs. 11B(G–O)). Collectively, these findings indicate that oxysterol-generating enzymes and their downstream oxysterol species are necessary and sufficient to mediate immune activation of NCOA7-deficient lung endothelium.

[0151] Deficiency of NCOA7 and intratracheal delivery of 7HOCA exacerbate PAH in vivo: To determine whether the presence of NCOA7 protects from EC immune activation in PAH severity, as a model of vascular proliferative PAH, Il6 transgenic (Tg + ) mice were used to induce severe pulmonary inflammation (M. K. Steiner et al., Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104, 236-244, 228p following 244 (2009)). Systemic knockout mice of NcoA7 were crossed with Il6 Tg+ Mice were mated to examine whether the loss of NCOA7 worsens the PH index in vivo (Figure 14(A)). Echocardiographic evaluation excluded significant changes in left ventricular function, as indicated by the left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and left ventricular posterior wall distances in diastole and systole (LVPW;d and LVPW;s) (Figure 13A(A - E)).

[0152] Ncoa7 null mice showed increased CH25H expression in the pulmonary artery, accompanied by increased plasma levels of 7HOCA and tetrol species (Figure 14(B, C, G) and Figure 13(F - I)). These findings were consistent with our studies of the plasma signatures of oxysterols and bile acids associated with PAH severity in humans and cultured PAECs. The increase in 7HOCA in Ncoa7 - deficient mice resulted in endothelial immune activation, as indicated by the promotion of VCAM1 expression and CD11b + monocyte infiltration (Figure 14(B - E)). Furthermore, Ncoa7 null mice showed increased pulmonary artery muscularization (Figure 14(F)), accompanied by a deterioration of the PAH hemodynamics with increased right ventricular systolic pressure (RVSP) and the Fulton index, an indicator of right ventricular remodeling (Figure 12A(B, I)).

[0153] Finally, to directly evaluate the pathogenicity of 7HOCA, a chronic hypoxia mouse model was used to continuously deliver either saline or 7HOCA intratracheally (Figure 14(J)), and echocardiography confirmed no changes in left ventricular function (Figure 13(J - N)). As expected, intratracheal delivery of 7HOCA upregulated endothelial immune activation, as indicated by enhanced VCAM1 expression and CD11b + monocyte infiltration (Figure 14(K - M)). Consistent with the gene knockout of Ncoa7, 7HOCA worsened PAH, as reflected by increased pulmonary artery remodeling and increased RVSP (Figure 12B(N, P)). Collectively, our data demonstrated that genetic deficiency of Ncoa7 or direct delivery of the inflammatory sterol 7HOCA is sufficient to promote PH in vivo.

[0154] The intronic SNP rs11154337 controls the binding of RelA / p65 to the non-canonical promoter of NCOA7 and is associated with the severity and mortality of PAH disease. From these data, so far, NCOA7 has been revealed to be a homeostatic brake that maintains lysosomal activity and sterol transport in order to attenuate the immune activation of ECs in PAH in inflammatory pathologies. Based on the role of single nucleotide polymorphisms (SNPs) in the regulation of gene promoter activity, we sought to investigate whether pathogenic NCOA7 deficiency could occur through genetic SNP-dependent control of NCOA7 expression and its downstream functions. First, we investigated the annotated SNPs based on their proximity to canonical and non-canonical promoters and the high levels of epigenetic marks indicating increased transcriptional activity. As a result, we found a candidate SNP, rs11154337, located in the vicinity of the intronic region close to the non-canonical promoter of NCOA7 and with a substantial load of histone modifications (W. J. Kent et al., The human genome browser at UCSC. Genome Res 12, 996-1006 (2002)). Second, since tandem regulation of both the short and long isoforms of NCOA7 is seen in PAH (Figure 4), we sought to identify the potential regulatory function of this SNP by positional backfolding to the canonical promoter. For this purpose, we used publicly available high-throughput chromatin conformation capture (3C) for human umbilical vein endothelial cells (GEO IDs GSM3438650 and GSM3438651) of the 3D-genome Interaction Viewer & database (3DIV) (D. Yang et al., 3DIV: A 3D-genome Interaction Viewer and database. Nucleic Acids Res 46, D52-D57 (2018)). Such maps revealed that the SNP rs11154337 has long-range interactions with the canonical promoter region of NCOA7 exceeding 120 kilobases (Figure 15A(A)).To establish this interaction, the inventors performed 3C in human PAEC and found that the 3′ end of the DNA segment digested with a restriction enzyme containing the NCOA7 transcription start site (N3) ligated to the 5′ end of the digested segment containing the SNP rs11154337 (S5) to produce a nucleotide fusion PCR product (N3S5) (confirmed by sequencing) (Figure 15A (B, C)). Furthermore, ChIP against RelA / p65 revealed significant enrichment for the region containing the SNP, indicating the presence of a p65 protein-SNP complex (Figure 15A (D)).

[0155] In combination with the finding that NCOA7 controls an oxysterol signature associated with PAH severity and mortality, and given that the SNP rs11154337 of the NCOA7 gene controls the composition of the NCOA7 gene, we sought to examine whether this SNP is also associated with disease severity and mortality using two independent PAH cohorts (Figure 12a (a - c)). First, a single - center PAH cohort of European - descent subjects from the University of Pittsburgh Medical Center (UPMC, N = 93) was analyzed. The G allele was found to be associated with a significant improvement in 6 - minute walk distance (P = 0.0130; β = 66.90, 95% CI [14.45 - 119.36]) (Figure 12A(A)). Importantly, after adjustment for age, gender, and use of vasodilators, patients with the homozygous G allele had a significantly prolonged survival (P = 0.0250; hazard ratio = 0.44, 95% CI [0.21 - 0.90], Figure 12A(B)). Second, a multi - center PAH cohort of European - descent from the Sitaxsentan To Relieve Impaired Exercise (STRIDE) trial, which included 45 pulmonary hypertension centers in the United States and Canada (STRIDE, N = 63 (R. L. Benza et al., Endothelin - 1 Pathway Polymorphisms and Outcomes in Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 192, 1345 - 1354 (2015))), the presence of the G allele was verified to confer a survival benefit (P = 0.0002, hazard ratio = 0.49, 95% CI [0.34 - 0.71], Figure 12B(C)). Thus, analysis of genomic, metabolomic, and clinical datasets across PAH patient cohorts suggested a correlative activity between NCOA7 and SNP rs11154337 and glucuronidated oxysterols and the clinical outcome of PAH.

[0156] SNP rs11154337 regulates NCOA7 and its downstream pathogenic functions: Using this concept and guided by the fact that the G allele of the NCOA7 intron SNP rs11154337 has a negative correlation with both the oxysterol signature predicting mortality and the clinical indicators of PAH, we sought to determine whether this SNP controls the expression of NCOA7, lysosomal activity, and the production of oxysterol and bile acid metabolites to regulate the behavior of ECs. To study the cellular and biological activities of SNP rs11154337 embedded near the non-canonical NCOA7 promoter, a set of genetically matched isogenic induced pluripotent stem cell (iPSC) lines were generated using the allelic variants of SNP rs11154337 via CRISPR-Cas9 gene editing (C / C vs C / G genotype, Figures 12A(D) and 15A(F)). Subsequently, the iPSCs were differentiated into ECs (iPSC-ECs) and purified by magnetic separation based on vascular endothelial cadherin (VE-Cadherin; also known as CD144) (M. Gu, Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Cells. Curr Protoc Hum Genet, e64 (2018)). The purified iPSC-ECs showed significant enrichment of the EC markers CD34, CD144, and CD309, and immunofluorescent staining of iPSC-ECs for CD144 and CD31 revealed patterning consistent with endothelium (Figure 15B(G,H)). Furthermore, the iPSC-ECs exhibited angiogenic ability as observed by angiogenesis in Matrigel lacking growth factors (Figure 15B(H)).

[0157] C / G iPSCs-ECs had higher expression of both short and long NCOA7 isoforms compared to C / C strains, and it was confirmed that the G allele increased NCOA7 transcription (Figure 12A (E, F)). An inferred explanation for the long-range regulation of SNP rs11154337 and the canonical promoter of NCOA7 was provided, and from prior chromatin capture data, an interaction between SNP rs11154337 and the canonical promoter in human umbilical vein endothelial cells was demonstrated (Figure 15A (A-D)). Consistent with the observed differences in NCOA7 expression and our prior findings using NCOA7 knockdown, lysosomal activity, sterol homeostasis, and immune activation in iPSCs-ECs were allele-dependent. iPSCs-ECs with the G allele, i.e., high NCOA7 expression, showed an increase in its binding partner ATP6V1B2, followed by a decrease in lysosomal pH, as indicated by the attenuation of SiR-lysosome cleavage (Figure 12A (G-I)). Furthermore, the presence of the G allele prevented lysosomal hypertrophy compared to the isogenic C allele iPSC-EC strain, indicating proper lysosomal acidification and consequent maintenance of sterol homeostasis (Figure 12B (J) and Figure 15B (I,)).

[0158] Similar to RNAi against NCOA7, the isogenic C allele strain had low NCOA7 expression, resulting in higher sterol content and higher expression of CH25H and its downstream metabolite 25HC (the enzyme responsible for the production of downstream oxidized species such as 7HOCA) (Figure 12B (K-O)). Increased production of 7HOCA led to increased endothelial immune activation in the isogenic C allele iPSC strain, as reflected by increased expression of VCAM1 and enhanced immune cell adhesion (Figure 12B (P-S)). Thus, consistent with the relevance of the G allele of SNP rs11154337 as a protective factor against oxysterol production and PAH severity, the G allele increased NCOA7 expression and enhanced the regulation of lysosomal acidification, oxysterol production, and consequent EC immune activation downstream.

[0159] Structural modeling and molecular simulations identify novel therapeutic activators of NCOA7: To identify small-molecule activators of NCOA7, structure-based calculations consisting of three parts, druggability simulation, pharmacophore modeling, and virtual screening, were performed (Figure 16A (A-C)). Druggability simulation was carried out in the presence of probe molecules representing explicit water and drug-like fragments using the modeled structure of NCOA7. As probe molecules, acetamide, acetate, benzene, imidazole, isobutane, isopropanol, and isopropylamine were used, and six independent simulations of 40 ns each were performed. A molecular pocket was identified in three runs by the high affinity for binding to the probe molecules (Figure 16A (A), cyan spheres). This site also shows the hinge residues obtained from the Gaussian Network Model (GNM) analysis of NCOA7 (Figure 17 (A)). The hinge residues in or near the binding pocket are L83 (mode 1), L72 (mode 2), and E66 and W81 (mode 3) (Figure 17 (B)). In particular, the hinge site has been shown in previous studies to play an important role in mediating the functional dynamics of proteins and is therefore used as a target site for binding small-molecule modulators of protein function. For these reasons, the identified molecular pocket was selected for further analysis using pharmacophore modeling enhanced by both druggability simulation and GNM analysis.

[0160] Using the inventors' tool Pharmmaker, pharmacophore modeling was performed, and then the high-affinity residues of the identified molecular pocket (i.e., N62, E66, P80, and W81; Figure 16A(B)) were selected (J. Y. Lee, et al., Pharmmaker: Pharmacophore modeling and hit identification based on druggability simulations. Protein Sci 29, 76-86 (2020)). The interactions of these residues with the probe molecules were ranked based on their frequency of occurrence during the simulation. In particular, among the probe molecules, two benzenes showed a high interaction tendency with N62, P80, and W81, and one isopropylamine interacted with E66 (Figure 16A(B), black is benzene, blue is isopropylamine). Using molecular dynamics (MD) snapshots that simultaneously display multiple frequently observed (i.e., entropically favorable) interactions, a pharmacophore model composed of one hydrogen bond donor, three hydrophobic rings, and two aromatic rings was constructed (Figure 16A(C)).

[0161] Next, the pharmacophore model was screened with the MolPort small molecule library via the ZINC (T. Sterling, J. J. Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)) and Pharmit (T. Sterling, J. J. Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)) servers to obtain an ensemble of compounds. The top-scoring compounds were selected as hits and further experimental verification was performed. For compound MolPort-004-267-958 (referred to as 958 herein), preliminary data suggesting that it is an activator of NCOA7 were obtained compared to other predicted compounds. Next, the inventors attempted to further investigate the binding behavior of 958 by MD simulation.

[0162] An analog 958 of the activator 958 with stronger binding affinity was obtained by the improvement of the activator 958 ami : To create a molecule that binds with high avidity to the molecular pocket of NCOA7, MD simulations were performed. Therefore, a 0.6 μs MD simulation of all atoms of 958 (or three independent 0.2 μs runs) was carried out to clarify the most important functional groups and interaction characteristics. Based on these simulations, an analog of 958 was designed, and the O 15 atom of the ester functional group was replaced with N 15 -H to create an amide functional group, and the resulting compound is referred to herein as 958 ami (Figure 16A (D, E)). Subsequently, MD simulations using 958 ami further clarified the enhancement of its activity. To compare the parent structure and the maintained structure, nine residues with high interaction affinity with the compound were selected: H56, N62, I65, E66, A69, R70, Q77, G78, and W81. The overall contact time of these residues was longer than 0.35 μs out of 0.6 μs of 958 or 958 ami (Figure 18 (A - C)). For N62 - O ami , E66 - O 10 / O 10 / O 11 , and W81 - N 15 / O 17 of 958, H56 - O1 of 958 formed a strong interaction defined as a contact time exceeding 0.3 μs (Figure 16A (D); orange solid line). In particular, atoms 16 to 34 had no significant interaction with the NCOA7 binding pocket. However, when O 15 was replaced with N 15 -H, multiple strong interactions occurred with many residues within the pocket. Furthermore, the substitution at N 15 showed strong interactions with I65 and W81, caused new interactions between atoms 16 to 34 and A69, R70, and Q77, and C 23 / C 24 -G78 and O 10 / O 11-N62 / E66 interaction was enhanced. Finally, using hydrogen bond analysis with a cutoff distance of 3.0 Å and an angle of 160° between the donor and acceptor atoms, it was identified that E66 has a significantly higher tendency to form hydrogen bonds with 958 ami than the parent compound.

[0163] When the binding affinity was calculated using PRODIGY-LIG (T. Sterling, J. J. Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)), it was revealed that 958 ami binds more stably to NCOA7 than the parent compound 958 (Figure 19(A)). The corresponding binding pose at 100 - 200 ns had a binding affinity of -8.16 ± 0.16 kcal / mol (Figure 16A(E), Example 1). 958 ami also had two additional stable poses, one where W81 lost its interaction with N 15 (Figure 19(B), Example 2, -7.71 ± 0.16 kcal / mol), and the other where the compound rotated upside down in the pocket (Figure 19(B), Example 3, -8.83 ± 0.19 kcal / mol).

[0164] Administration of the NCOA7 activator 958 ami reverses the disease in the PAH model: To evaluate the downstream molecular functions of NCOA7 activation by 958 ami , a proximity ligation assay was performed to evaluate the interaction between ATP6V1B2 and NCOA7. As expected, the application of 958 ami significantly induced the number of amplifications per cell, suggesting molecular enhancement at the lysosome level (Figure 16B(F, G)). Maintenance of lysosome acidification by 958 ami similarly inhibited the induction of CH25H under IL-1β, which in turn corresponded to a decrease in EC immune activation as shown by VCAM1 expression and immune cell adhesion to the monolayer (Figure 16B(H - L)).

[0165] Next, 958ami We sought to determine whether it protects against endothelial immune activation in an inflammatory monocrotaline PAH rat model. Rats were intraperitoneally injected with DMSO or 958 ami (7.5 mg / kg) for 10 days after monocrotaline loading (Figure 16B(M)). ami Rats treated with 958 ami showed no significant hepatotoxicity or nephrotoxicity, nor changes in left ventricular function compared to vehicle controls (Figure 20(B - K)). Rats treated with the NCOA7 activator 958 + showed decreased CH25H expression, with a corresponding attenuation of VCAM1 expression in endothelium and CD11b ami monocyte infiltration in pulmonary vessels (Figure 16B and 16C(N - Q)). As a result, the pulmonary vessels showed a decrease in muscularization, which corresponded to a significant decrease in both right ventricular hypertrophy and RVSP (Figure 16C(R - T)). Overall, these data identified 958

[0166] as a novel therapeutic agent that reverses PAH pathology potentially associated with immunoregulatory disorders.

[0166] Using large - scale multi - dimensional genomic and metabolomic analyses and attendant mechanistic experiments, it was found that NCOA7 regulates lysosomal activity and EC sterol metabolism to function as a homeostatic brake, preventing oxysterol - induced inflammation, EC dysfunction, and PAH. Most notably, the presence of the G allele at SNP rs11154337 enhances NCOA7 expression, thereby reducing inflammation in PAH, establishing a fundamental genetic association between SNP rs11154337 and PAH mortality, and a mechanistic proof of the metabolomics association between the oxysterol signature and PAH severity. Finally, this study establishes a new paradigm linking basic lysosomal biology and oxysterol metabolism to EC behavior, with broad implications for the development of molecular diagnostics and therapies in PAH and other inflammatory vascular diseases.

[0167] The identification of NCOA7 as a major regulator of PAHs has broad implications for human diseases. The role of NCOA7 in immune regulation described herein is thought to indicate a much broader role of NCOA7 isoforms and related proteins. Recent studies have reported unique activities of the short isoforms of NCOA7, but the findings described herein indicate additive or synergistic behavior of both isoforms in regulating EC inflammation via oxysterols. NCOA7 isoforms have Tre2 / Bub2 / Cdc16 (TBC), lysine motif (LysM), and domain catalytic (TLDc) domains. All proteins containing TLDc physically interact with V-ATPase, thereby defining a new class of V-ATPase regulatory proteins.

[0168] At the cellular level, this study highlights the extensive role of lysosomes in EC function and PAH. Previous clinical findings have suggested a relationship between rare, recessive loss-of-function lysosomal storage disorders and pulmonary vascular disease. For example, human mutations in various V-ATPase subunits (e.g., ATP6V1A and ATP6V1E1) may present with pulmonary artery stenosis or hypoplasia and right ventricular hypertrophy (T. Van Damme et al., Mutations in ATP6V1E1 or ATP6V1A Cause Autosomal-Recessive Cutis Laxa. Am J Hum Genet 100, 216-227 (2017)). PAH has also been observed in mucolipidosis, a disease caused by lysosomal enzyme processing dysfunction. High pulmonary artery pressure has been reported in patients with Gaucher disease, a disease caused by a deficiency of lysosomal β-glucosidase (Figure 6B (F, G)), and is known to be associated with Group V PH. Furthermore, Niemann-Pick disease and Fabry disease present with severe lung dysfunction and often coexist with Group III PH. These rare genetic diseases more broadly and clearly demonstrate the causal relationship between lysosomal dysfunction and PAH. In fact, given the average C allele frequency of SNP rs1115447 in the world's populations (approximately 0.48 - 0.52), approximately one-quarter of PAH patients with the C / C genotype are expected to suffer from a worsening mortality rate. Furthermore, based on the previously reported principle of multiplicative heterozygosity for BMPR2 mutations in familial PAH, it is not yet known whether the worsening of PAH or other lysosomal storage disorders may be even greater in carriers of one allele of a known familial PAH mutation or lysosomal enzyme mutation when accompanied by the SNP rs1115447 C / C genotype.

[0169] In summary, by combining multi-dimensional analysis of genomic and metabolome datasets with in vitro and in vivo mechanistic validation, we defined the essential and SNP-dependent role of NCOA7 and the control of lysosomal activity and sterol homeostasis to alleviate inflammation, EC dysfunction, and PAH. In the computational modeling of the molecular pocket of NCOA7, compound 958 ami and its derivatives were developed. In vitro and in vivo studies revealed that compound 958 ami hinders oxysterol-mediated endothelial immune activation and reverses the disease in monocrotaline rats with PAH, and that a treatment such as 958 ami would be a compelling complement to existing vasodilatory therapies and other disease-modifying drugs in development that primarily target cell proliferation and survival pathways.

[0170] Example 2: In vitro assay of viruses SNP rs11154337 and 958 regulate the entry of various pseudotyped enveloped viruses. As shown in Figure 21, iPSCs transfected with the human ACE2 receptor showed a decrease in the entry of multiple pseudotyped coronaviruses, and herpesviruses transfected with the human ACE2 receptor and having the G allele at SNP rs11154337 showed a decrease in the entry of multiple pseudotyped coronaviruses, and herpesviruses having the G allele at SNP rs11154337 increased the expression of NCOA7. Furthermore, when the NCOA7 activator 958 was applied, the activity of NCOA7 that prevents pseudotyped SARS-CoV-2 infection (D614G spike) was enhanced in HEK293 cells transfected with the human ACE2 receptor. In iPS cells transfected with the human ACE2 receptor, 958 parent (2 μM) and 958 ami (5 μM) decreased BA.2 spike pseudotyped virus infection. Also, 958 parent (2 μM) and 958 ami (5 μM) decreased D614G spike pseudotyped virus infection in iPSCs transfected with the human ACE2 receptor.

[0171] Example 3: SARS-CoV2 in vivo assay Referring to Figure 22, coronavirus-infected human ACE2 transgenic mice treated with the NCOA7 activator 958 had reduced lung inflammation and mortality. Human ACE2 transgenic mice infected with two different coronavirus strains showed a significant improvement in mortality when treated with 958. Furthermore, mouse lung tissue showed a decrease in viral load and attenuation of various inflammatory markers (IL-1α, IL-1β, IFN-γ, VCAM1, and ICAM1) when treated with 958 via RT-qPCR.

[0172] Example 4: Reduction of lung inflammation in bacterial infection in vivo Mice were infected with Klebsiella pneumoniae. Referring to Figure 23, these mice showed improvement in acute lung injury when treated with the NCOA7 activator 958. (Figure 23(A, B) Representative images of lung tissue sections stained with H&E 48 hours after intratracheal infection with Klebsiella pneumoniae in mice treated with DMSO compared to those treated with 958 (5 mg / kg IP daily for 3 days). Mice treated with 958 had reduced inflammation as shown by ELISA of lung homogenates for the inflammatory cytokines IL-1β, TNF-α, and IL-6 (Figure 23(C)-(E)).

[0173] Example 5 - Stroke Deficiency of Ncoa7 worsens survival in a mouse model of ischemic stroke. Referring to Figure 24, wild-type mice and Ncoa7-deficient mice were subjected to an experimental model of ischemic stroke. Mice were subjected to sham surgery or transient middle cerebral artery occlusion (tMCAO) for 60 minutes until cerebral blood flow recovered. Thereafter, the mice were harvested at 24 hours and 48 hours for further study. It was found that Ncoa7 knockout mice had significantly lower survival rates at 24 hours and 48 hours after tMCAO compared to wild-type controls. There were no significant changes in body weight at 24 hours and 48 hours after tMCAO. Using a laser Doppler flowmeter, regional cerebral blood flow (rCBF) in the contralateral (CL) and ipsilateral (IL) cerebral cortex of the mice was quantified. Ncoa7-deficient mice had increased rCBF in the IL cerebral cortex at 24 hours after tMCAO compared to wild-type controls.

[0174] Deficiency of NCOA7 increases infarct volume and capillary leakage in mice after ischemic stroke. As shown in Figure 25, mouse brain tissue was sectioned into 30-micron slices and then immunofluorescent staining was performed. The brain sections were stained with microtubule-associated protein 2 (MAP2), a neuronal marker, to evaluate the infarct volume after tMCAO. In NCOA7-deficient mice, the infarct volume after ischemic stroke was significantly increased, indicating greater neuronal death. There were no significant changes in tissue swelling compared to wild-type controls. To evaluate capillary leakage, the brain sections were stained with plasma protein albumin. In particular, in NCOA7-deficient mice, there was a tendency for greater leakage of albumin into the brain tissue after tMCAO compared to wild-type controls, indicating greater damage to the cerebral microvasculature.

[0175] Deficiency of NCOA7 results in exacerbation of neuroinflammation after ischemic stroke. As shown in Figure 26, brain sections were stained using an immunofluorescence protocol. The brain was stained with glial fibrillary acidic protein (GFAP), an astrocyte marker, ionized calcium-binding adapter molecule 1 (IBA1), a microglia marker, and a nuclear marker (DAPI). Images were obtained using a confocal microscope in both the cerebral cortex (Ctx) and striatum (Str) of wild-type mice and Ncoa7 knockout mice after tMCAO. In NCOA7-deficient mice, neuroinflammation was significantly exacerbated, as indicated by the swelling of astrocytic processes, in both tissues of the cerebral cortex and striatum. Furthermore, microglia were hypertrophied and elongated in knockout mice compared to wild-type controls. These data indicate substantial reactive gliosis and neuroinflammation in the Ncoa7-deficient brain after tMCAO.

[0176] Deficiency of NCOA7 results in prominent myelin hyperplasia in a mouse model of ischemic stroke. Referring to Figure 27, brain sections were stained with myelin basic protein (MBP) using an immunofluorescence protocol. NCOA7-deficient mice had overall significantly enhanced myelination, particularly at the level of the corpus callosum (CC) and external capsule (EC). In particular, no significant difference was observed in the comparison between the contralateral hemisphere (CL) and ipsilateral hemisphere (IL) after tMCAO.

[0177] The present invention has been described with reference to specific exemplary embodiments. However, it will be recognized by those skilled in the art that various substitutions, modifications, or combinations of any of the exemplary embodiments can be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is not limited by the description of the exemplary embodiments, but rather by the scope of the originally filed appended claims.

Claims

1. A pharmaceutical composition for treating inflammation in a patient, comprising: 【Chemistry 1】 [Wherein, R １１ , １－３ , １０ , ７ , １－３ , ６ , ９ , １－３ , ８ , １－３ , １１ , ８ , ７ , １２ , １－３ , １－３ , ６ , １０ , ５ , ９ , １２ , １－３ and R ２ are each independently -H or -C １－３ alkyl; Z is O or NH; X １ , X ２ , X ３ are each independently N or C; X ４ is ortho, meta or para to X １ and is N or C; Y ２ is -H, -C １－３ alkyl, halo, or -NO ２ ; Y １ is -H, -C １－３ alkyl, halo, -NO ２ , -CN, -CF ３ , -SO<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Compounds having; or MolPort-005-950-209; MolPort-005-043-754; MolPort-044-323-945 (ZINC581791018); MolPort-044-179-284; MolPort-006- 808-904; MolPort-002-633-931 (ZINC9015186); MolPort-004-932-049 (ZINC9050354); MolPort-006-808-656 (ZINC9059787); M olPort-002-613-702; MolPort-004-267-958; MolPort-004-509-205; MolPort-001-015-690; MolPort-004-974-660; ZINC952864645; ZINC11785026; ZINC585262189; ZINC4026555; ZINC169785251; ZINC275180256; ZINC652604, or; one or more of ZINC9583892, or A pharmaceutical composition comprising a pharmaceutically acceptable salt in an amount effective in reducing inflammation in a patient.

2. The pharmaceutical composition according to claim 1, wherein the inflammation is cardiovascular inflammation or vasculitis, or is associated with diseases such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, viral infection, bacterial infection, fungal infection, parasitic infection, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegeneration, cancer, autoimmune disease, or innate and adaptive immune diseases.

3. The pharmaceutical composition according to claim 1, wherein the patient has vascular inflammation.

4. The pharmaceutical composition according to claim 1, wherein the inflammation is related to a viral or bacterial infection.

5. The pharmaceutical composition according to claim 2, wherein the inflammation is related to the patient's coronavirus infection.

6. The pharmaceutical composition according to claim 5, wherein the inflammation is related to severe acute respiratory syndrome caused by a coronavirus infection in the patient.

7. The pharmaceutical composition according to claim 2, wherein the inflammation is related to a bacterial infection in the patient.

8. The pharmaceutical composition according to claim 7, wherein the bacterial infection is a Klebsiella pneumoniae infection in the patient.

9. The pharmaceutical composition according to claim 1, wherein the inflammation is related to one or more of the following: pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease, pulmonary hypertension due to chronic pulmonary thrombosis, and pulmonary hypertension of unknown cause.

10. The pharmaceutical composition according to claim 1, wherein Z is NH.

11. R １ and R ２ The pharmaceutical composition according to claim 1, wherein is independently Me or H.

12. The pharmaceutical composition according to claim 1, wherein the compound is compound 958 (MolPort-004-267-958) or a pharmaceutically acceptable salt thereof.

13. The above compound has an exemplary structure: 【Chemistry 2】 Compound 958 having ａｍｉ The pharmaceutical composition according to claim 1, or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical composition according to claim 1, administered to the patient in an amount of 1 μg to 10 g or 1 ng to 100 mg / kg of the compound per day, or up to a concentration in the patient's body fluids ranging from 1 to 40 μM.

15. The pharmaceutical composition according to claim 1, wherein the patient is heterozygous or isozygous with respect to C of rs11154337.

16. The pharmaceutical composition according to claim 15, wherein, prior to administration, the patient's genetic data is obtained and it is determined whether the patient has one or two alleles with respect to rs11154337C.

17. The pharmaceutical composition according to claim 6, wherein the coronavirus infection is one or more of the following diseases caused by the same: Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), or Coronavirus Disease 2019 (COVID-19).

18. The pharmaceutical composition according to claim 17, wherein the coronavirus is SARS-CoV-2.

19. The pharmaceutical composition according to claim 1 for reducing the infectivity of coronavirus infection or herpesvirus infection in cells.

20. structure: 【Transformation 3】 [In the formula, R １ and R ２ These are independently -H or -C １－３ It is alkyl; Z is O or NH; X １ , X ２ , X ３ Independently, N or C; X ４ is, X １ It is ortho, meta, or para, and N or C; Y ２ is -H, -C １－３ Alkyl, halo, or -NO ２ Y １ is -H, -C １－３ Alkyl, Halo, -NO ２ -CN, -CF ３ , -SO ２ R ４ (Here, R ４ is -OH, -C １－３ Alkyl), -NHR ５ (Here, R ５ is H or -C １－３ Alkyl), -NHR ６ (Here, R ６ is -H or C １－３ (It is alkyl), -NHC(O)-R ７ (Here, R ７ is -H or -C １－３ (It is alkyl), -OR ８ (Here, R ８ is -H or -C １－３ (It is alkyl), -OC(O)-R ９ (Here, R ９ is -H or -C １－３ (It is alkyl), -C(O)-R １０ (Here, R １０ is -H or -C １－３ (It is alkyl), or -C(O)-R １１ -R １２ (Here, R １１ is O or NH, R １２ -H, -C １－３ (It is alkyl.) Compounds comprising the above, or pharmaceutically acceptable salts thereof (excluding MolPort-004-267-958).

21. The compound according to claim 20, wherein Z is NH.

22. R １ and R ２ The compound according to claim 20 or 21, wherein is independently Me or H.

23. R １ and R ２ is H, a compound according to claim 20 or 21.

24. structure: 【Chemistry 4】 A compound according to claim 20, or a pharmaceutically acceptable salt thereof, having the above.

25. A pharmaceutical composition comprising a gene editing tool for treating a patient having SNP rs11154337 with C, wherein the patient has inflammation and / or an inflammatory disease, or is associated with a disease such as pulmonary hypertension, restenosis, essential hypertension, atherosclerosis, viral infection, bacterial infection, fungal infection, parasitic infection, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegeneration, cancer, autoimmune disease, or innate and adaptive immune disease.

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