Methods and compositions for treating inflammation and inflammatory conditions

Abstract

The present invention provides methods of treating a patient (e.g., a human patient) suffering from inflammation or an inflammatory disease or a disease in which inflammation is present, such as cardiovascular or vascular endothelial inflammation, such as pulmonary arterial hypertension, restenosis, primary hypertension, atherosclerosis, stroke, sepsis or a viral infection (e.g., coronavirus infection, e.g., SARS-CoV-2). The method comprises administering to the patient an amount of a compound described herein effective to treat the patient. NCOA7 activating compounds and compositions are also provided. Also provided are gene editing methods for reducing inflammation or inflammatory conditions in a patient having C at SNP rs11154337, comprising editing C to G.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 346,453 filed on May 27, 2022 and U.S. Provisional Patent Application No. 63 / 502,499 filed on May 16, 2023, the disclosures of which are hereby incorporated by reference in their entirety.

[0003] Statement Regarding Federal Funding

[0004] This invention was made with government support under Grant No. HL 138437 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] The sequence listing associated with this application is submitted in electronic form through the Patent Center and is hereby incorporated by reference in its entirety into the 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.

[0006] Inflammation is an important component of many diseases, including vascular and heart diseases, but also non-vascular diseases such as sepsis, COVID (coronavirus disease), ARDS (acute respiratory distress syndrome), acute lung injury, stroke, neurodegenerative diseases, cancer and autoimmune diseases. In the example of vascular inflammation, it regulates key vascular and endothelial pathological phenotypes (e.g., in atherosclerosis, essential hypertension, peripheral vascular disease and restenosis, etc.), but its exact pathogenic mechanism remains a mystery, especially in pulmonary vascular diseases, such as arterial pulmonary hypertension (PAH), or other forms (grades 2-5) of pulmonary hypertension. In one example, PAH includes the following categories: idiopathic PAH, hereditary PAH (e.g., BMPR-2, etc.), diseases such as PAH associated with connective tissue disorders, HIV infection, portopulmonary hypertension, congenital heart disease, schistosomiasis and hemolytic anemia, pulmonary veno-occlusive disease and persistent pulmonary hypertension of the newborn.

[0007] Identifying active pharmaceutical ingredients (e.g., drugs) that can reduce inflammation (e.g., vascular or endothelial inflammation) would provide useful therapies for treating a variety of diseases, such as treating inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with vascular endothelial inflammation as a symptom, such as pulmonary hypertension (PH, including PH types of grades 1-5, such as arterial pulmonary hypertension (PAH), pulmonary hypertension caused by left heart disease, pulmonary hypertension caused by lung disease, pulmonary hypertension caused by chronic thrombosis of the lung, and pulmonary hypertension of unknown etiology), restenosis, essential hypertension, atherosclerosis, and stroke), diseases characterized by vascular inflammation, or innate and acquired immune diseases such as autoimmune diseases, heart disease, lung disease, sepsis, cancer, and neurodegenerative diseases, such as inflammation associated with, for example, heart disease, lung disease, sepsis, cancer, and neurodegenerative diseases. It is desirable to identify such useful active pharmaceutical ingredients and related methods of use. Summary of the invention

[0008] A method for treating inflammation in a patient (e.g., a human patient) is provided. The method comprises administering to the patient an amount of a compound having the following structure in an amount effective to reduce inflammation in the patient:

[0009]

[0010] Wherein 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (wherein R4 is -OH or -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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 alkyl), -OC(O)-R9 (wherein R9 is -H or -C 1-3 alkyl), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 -H or -C1-3 alkyl); or

[0011] 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) ; MolPort-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 ZINC9583892,

[0012] or a pharmaceutically acceptable salt thereof.

[0013] Provided are methods of treating pulmonary arterial hypertension (PAH) in a patient, comprising administering to the patient a compound having the following structure:

[0014]

[0015] Wherein 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H or -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (wherein R4 is -OH or -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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 Alkyl; C 1-3Alkoxy), -OC(O)-R9 (wherein R9 is -H or -C 1-3 Alkyl; ester), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 -H or -C 1-3 alkyl); or

[0016] 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) ; MolPort-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 ZINC9583892,

[0017] or a pharmaceutically acceptable salt thereof.

[0018] Compounds are provided comprising the structure:

[0019]

[0020] in

[0021] 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (where R4 is -OH, -C 1-3alkyl), -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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 alkyl), -OC(O)-R9 (wherein R9 is -H or -C 1-3 alkyl), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 -H, -C 1-3 Alkyl), or a pharmaceutically acceptable salt thereof, excluding MolPort-004-267-958.

[0022] A pharmaceutical composition is provided, comprising:

[0023] A compound that either:

[0024] Has the following structure:

[0025]

[0026] Wherein R1 and R2 are both or individually (independently) -H or -C 1-3 alkyl; Z is O or NH; X1, X2, X3 are independently N or C; X4 is located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen (-F, -Cl, -Br or -I) or -NO2 (nitro); Y1 is -H, -C 1-3 Alkyl, halogen (-F, -Cl, -Br or -I), -NO2, -CN (nitrile), -CF3 (trifluoromethyl), -SO2R4 (wherein R4 is -OH, -C 1-3 alkyl), -NHR5 (wherein R5 is H, -C 1-3 Alkyl; sulfonyl), -NHR6 (R6 can be -H, -C 1-3 Alkyl; amino), -NHC(O)-R7 (R7 can be -H, -C 1-3 Alkyl; amide), -OR8 (R8 can be -H, -C 1-3 Alkyl; C 1-3 Alkoxy), -OC(O)-R9 (R9 can be -H, -C 1-3 Alkyl; ester), -C(O)-R 10 (R10 Can be -H, -C 1-3 alkyl; aldehyde or ketone) or -C(O)-R 11 -R 12 (R 11 Can be O or NH; R 12 Can be -H, -C 1-3 alkyl; amide or ester), or a pharmaceutically acceptable salt thereof; or

[0027] The compound is selected from one or more of the following: 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 (ZINC90 59787); MolPort-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 and ZINC9583892, or a pharmaceutically acceptable salt thereof; and

[0028] Pharmaceutically effective excipients,

[0029] The composition comprises an amount of the compound effective to treat or reduce inflammation, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with 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 in a patient (e.g., a human patient), or for treating coronavirus infection, such as SARS-CoV-2 infection, in a patient (e.g., a human patient).

[0030] Also provided is a method for treating a patient who has a C at SNP rs11154337 and suffers from inflammation and / or an inflammatory disease, such as cardiovascular inflammation or vascular inflammation, or the inflammation and / or inflammatory disease 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, neurodegenerative disease, cancer, autoimmune disease, or innate and acquired immune disease, the method comprising using gene editing, such as but not limited to CRISPR / Cas9 or TALEN-based methods, to change one or more Cs at SNP rs11154337 to G.

[0031] The following provide illustrative, non-limiting aspects, embodiments, or examples of the invention.

[0032] Clause 1. A method of treating inflammation in a patient (eg, a human patient), comprising:

[0033] An amount of a compound having the following structure is administered to the patient in an amount effective to reduce inflammation in the patient:

[0034]

[0035] Wherein 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (wherein R4 is -OH or -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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 alkyl), -OC(O)-R9 (wherein R9 is -H or -C 1-3 alkyl), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12-H or -C 1-3 alkyl); or

[0036] 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) ;MolPort-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;ZINC9583892 or one or more thereof,

[0037] or a pharmaceutically acceptable salt thereof.

[0038] Item 2. A method according to Item 1, wherein the inflammation is cardiovascular inflammation or vascular inflammation, 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, neurodegenerative disease, cancer, autoimmune disease, or innate and acquired immune diseases.

[0039] Clause 3. The method according to Clause 1, wherein the patient suffers from vascular inflammation.

[0040] Clause 4. The method according to Clause 1, wherein the inflammation is associated with a viral infection or a bacterial infection.

[0041] Clause 5. The method according to clause 2, wherein the inflammation is associated with a coronavirus infection in the patient, optionally associated with severe acute respiratory syndrome caused by a coronavirus infection in the patient.

[0042] Clause 6. The method according to Clause 2, wherein the inflammation is associated with a bacterial infection of the patient.

[0043] Clause 7. The method according to Clause 6, wherein the bacterial infection is a Klebsiella pneumoniae infection in the patient, optionally reducing or preventing lung damage in the patient.

[0044] Clause 8. A method according to clause 1, wherein the inflammation is associated with one or more of: pulmonary arterial hypertension (PAH), pulmonary hypertension caused by left heart disease, pulmonary hypertension caused by lung disease, pulmonary hypertension caused by chronic thrombosis in the lungs, and pulmonary hypertension of unknown etiology.

[0045] Clause 9. A method of treating pulmonary arterial hypertension (PAH) in a patient, comprising administering to the patient a compound having the following structure:

[0046]

[0047] Wherein 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H or -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (wherein R4 is -OH or -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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 Alkyl; C 1-3 Alkoxy), -OC(O)-R9 (wherein R9 is -H or -C 1-3 Alkyl; ester), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 -H or -C 1-3 alkyl); or

[0048] 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) ; MolPort-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 ZINC9583892,

[0049] or a pharmaceutically acceptable salt thereof.

[0050] Clause 10. The process according to any one of clauses 1 to 9, wherein Z is NH.

[0051] Clause 11. The process according to any one of clauses 1 to 10, wherein R1 and R2 are independently Me or H.

[0052] Clause 12. The method according to any one of clauses 1 to 10, wherein R1 and R2 are H.

[0053] Clause 13. The method according to any one of clauses 1 to 9, wherein the compound is compound 958 (MolPort-004-267-958), or a pharmaceutically acceptable salt thereof.

[0054] Clause 14. The method according to any one of clauses 1 to 9, wherein the compound is compound 958 ami , which has the following exemplary structure:

[0055]

[0056] or a pharmaceutically acceptable salt thereof.

[0057] Clause 15. The method according to any one of Clauses 1-14, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient in an amount effective to reduce inflammation in the patient or to treat pulmonary hypertension in the patient.

[0058] Item 16. The method according to any one of items 1-14, comprising administering to the patient daily 1 μg / kg to 10 g / kg, or 1 ng / kg to 100 mg / kg of the compound, or administering the compound to a concentration range of 1 to 40 μM in the patient's body fluids (e.g., blood, serum, plasma, etc.).

[0059] Clause 17. The method according to any one of clauses 1 to 16, wherein the patient is heterozygous or homozygous for C at rs11154337.

[0060] Clause 18. The method according to any one of clauses 1-16, wherein the patient is homozygous for C at rs11154337.

[0061] Clause 19. Method 17 or 18, further comprising obtaining genetic data of the patient and determining whether the patient has one or both alleles of C at rs11154337.

[0062] Clause 20. The method according to clause 17 or 18, further comprising determining whether the patient has one or both alleles that are C at rs11154337.

[0063] Clause 21. A method according to clause 5, wherein the coronavirus infection is one or more of: Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), or a disease caused thereby, such as Coronavirus Disease 2019 (COVID-19).

[0064] Clause 22. The method according to clause 21, wherein the coronavirus is SARS-CoV-2.

[0065] Item 23. The method according to Item 1, which is used to reduce the infectivity of a coronavirus or herpes virus infection in a cell.

[0066] Item 24. A compound comprising the structure:

[0067]

[0068] in

[0069] 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 located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H, -C 1-3 Alkyl, halogen, -NO2, -CN, -CF3, -SO2R4 (where R4 is -OH, -C1-3 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 1-3 alkyl), -OR8 (wherein R8 is -H or -C 1-3 alkyl), -OC(O)-R9 (wherein R9 is -H or -C 1-3 alkyl), -C(O)-R 10 (where R 10 -H or -C 1-3 alkyl) or -C(O)-R 11 -R 12 (where R 11 is O or NH and R 12 -H, -C 1-3 Alkyl), or a pharmaceutically acceptable salt thereof, excluding MolPort-004-267-958.

[0070] Clause 25. The compound according to clause 24, wherein Z is NH.

[0071] Clause 26. The compound according to clause 24 or 25, wherein R1 and R2 are independently Me or H.

[0072] Clause 27. The compound according to clause 24 or 25, wherein R1 and R2 are H.

[0073] Item 28. The compound according to Item 24, which has the following structure:

[0074]

[0075] or a pharmaceutically acceptable salt thereof.

[0076] Article 29. A pharmaceutical composition comprising:

[0077] A compound that either:

[0078] Has the following structure:

[0079]

[0080] Wherein R1 and R2 are both or individually (independently) -H or -C 1-3 Alkyl; Z is O or NH; X1, X2, X3 are independently N or C; X4 is located at the ortho, meta or para position of X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen (-F, -Cl, -Br or -I) or -NO2 (nitro); Y1 is -H, -C 1-3Alkyl, halogen (-F, -Cl, -Br or -I), -NO2, -CN (nitrile), -CF3 (trifluoromethyl), -SO2R4 (wherein R4 is -OH, -C 1-3 alkyl), -NHR5 (wherein R5 is H, -C 1-3 Alkyl; sulfonyl), -NHR6 (R6 can be -H, -C 1-3 Alkyl; amino), -NHC(O)-R7 (R7 can be -H, -C 1-3 Alkyl; amide), -OR8 (R8 can be -H, -C 1-3 Alkyl; C 1-3 Alkoxy), -OC(O)-R9 (R9 can be -H, -C 1-3 Alkyl; ester), -C(O)-R 10 (R 10 Can be -H, -C 1-3 alkyl; aldehyde or ketone) or -C(O)-R 11 -R 12 (R 11 Can be O or NH; R 12 Can be -H, -C 1-3 alkyl; amide or ester), or a pharmaceutically acceptable salt thereof; or

[0081] The compound is selected from one or more of the following: 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 (ZINC905 9787); MolPort-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 and ZINC9583892, or a pharmaceutically acceptable salt thereof; and

[0082] Pharmaceutically effective excipients,

[0083] The composition comprises an amount of a compound effective to treat or reduce inflammation, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with 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 in a patient (e.g., a human patient), or for treating a coronavirus infection, such as a SARS-CoV-2 infection, in a patient (e.g., a human patient).

[0084] Clause 30. The composition according to Clause 29, wherein Z is NH.

[0085] Clause 31. A composition according to clause 29 or 30, wherein R1 and R2 are independently Me or H.

[0086] Clause 32. A composition according to clause 29 or 30, wherein R1 and R2 are H.

[0087] Clause 33. The composition according to clause 29, having the following structure:

[0088]

[0089] or a pharmaceutically acceptable salt thereof.

[0090] Clause 34. The composition according to Clause 29, wherein the compound is MolPort-004-267-958.

[0091] Clause 35. A method for treating a patient who has a C at SNP rs11154337 and suffers from inflammation and / or inflammatory diseases, such as cardiovascular inflammation or vascular inflammation, or the inflammation and / or inflammatory diseases are associated with the following 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, neurodegenerative disease, cancer, autoimmune disease, or innate and acquired immune diseases, the method comprising using gene editing, such as but not limited to CRISPR / Cas9 or TALEN-based methods, to change one or more Cs at SNP rs11154337 to G. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 .Exemplary nucleic acid sequence (SEQ ID NO: 1).

[0093] Figure 2.An exemplary nucleic acid sequence of the rs11154337C allele [top strand, SEQ ID NO: 30].

[0094] FIG3 . Exemplary sequence of human NCOA7 mRNA (SEQ ID NO: 2).

[0095] Figure 4 .Convergent inflammatory regulation of NCOA7 in cellular, animal, and human examples of PAH. (A) Transcriptome analysis of human PAECs under control or IL-1β (N=3 / group). Z-scores are indicated as positive values ​​in blue and negative values ​​in gold. FDR-corrected P-values ​​for the listed genes were <0.05. (B and C) Expression of NCOA7 isoforms detected by RT-qPCR (N=3 / group). NCOA7 (red), CD31 in pulmonary vasculature of mice (N=8 / group) and human PH models (N=6-7 / group) + EC (green), α-SMA + (D and F) Immunofluorescence (IF) staining and (E and G) quantification of smooth muscle cells (white) and DAPI-stained nuclei (blue). (H and I) Violin plots of NCOA7-expressing ECs identified by single-cell RNA sequencing from lungs of controls or PAH patients (N>3 / group). Cells were identified as expressing NCOA7 if the converted expression value was >0.2. (J and K) NCOA7 isoform expression under RNAi for RELA (N=3 / group). Two-way ANOVA. (L) ChIP-qPCR for p65 / RelA binding to full-length and short-length promoter regions (N=3 / group). Unless otherwise stated, all data were analyzed by Student's t-test and are presented as mean ± SD.

[0096] Figure 5A and 5B .Inflammatory regulation of NCOA7 and in vitro tools to modulate its expression. (A) Structure of full-length and short-length isoforms of NCOA7 located at 6q22.33. Exons are represented by rectangles. Black rectangles represent the first exon of the short-length isoform. (B to E) Expression of NCOA7 isoforms in human PAEC detected by RT-qPCR (N=3 / group). (F and G) CD31 in whole lungs of IL-6 transgenic mice under chronic hypoxia detected by RT-qPCR + Expression of NCOA7 isoforms in isolated cells. (N=5-7 / group). (H to K) Expression of NCOA7 (red), CD31 in pulmonary vessels of mice (N=5 / group) and rat PH models (N=5 / group). + EC (green), α-SMA +Immunofluorescence staining and quantification of smooth muscle cells (white) and DAPI-stained nuclei (blue). (L to N) Expression of NCOA7 isoforms in HPAEC detected by RT-qPCR (N=3 / group). (O and P) Immunoblotting and densitometry of Myc-tagged NCOA7 isoforms (N=3 / group). Unless otherwise stated, all data were analyzed by Student's t-test and expressed as mean ± SD.

[0097] Figures 6A-6C .NCOA7 deficiency leads to lysosomal dysfunction and lipid accumulation under proinflammatory conditions. A) Transcriptome analysis of PAECs under IL-1β by RNAi against control or NCOA7 (N=3 / group). Z-scores are indicated as positive values ​​in blue and negative values ​​in gold. FDR-corrected P-values ​​of identified lysosomal genes were <0.05. (B) Expression of ATP6V1B2 under siNC or siNCOA7 detected by RT-qPCR (N=3 / group). (C) Expression of NCOA7 by lentiviral delivery of control (LV-GFP), NCOA7 短 or NCOA7 全长 ATP6V1B2 was expressed in 100% ATP-ATPase subunits (N=3 / group). Data were analyzed by one-way ANOVA. (D and E) Association of the V-ATPase subunit ATP6V1B2 with NCOA7 (orange) was measured by proximity ligation assay. The lower panel shows images of ATP6V1B2, NCOA7, or no antibody control. The upper panel shows images of ATP6V1B2 and NCOA7 antibodies with lentiviral-transduced GFP control, NCOA7 短 or NCOA7 全长Double incubation of . Quantification of amplified signals per cell (N = 5 cells / group). Data were analyzed by one-way ANOVA. (F and G) LysoLive probe (green) reflects β-galactosidase activity and thus lysosomal acidification. Quantified as fluorescence per cell (N = 10 cells / group). (H and I) Silicon rhodamine (SiR)-lysosomal dye targets active cathepsin D (purple) and indicates low lysosomal pH. Quantified as fluorescence per cell (N = 10 cells / group). (J) Live cell confocal microscopy of lysosomes (LysoTracker Deep Red) and lysosomal acidification (LysoSensor Green DND-189). (K) Ratio of median fluorescence intensity (MFI) of LysoSensor Yellow / Blue DND-160 dyes detected by flow cytometry, representing lysosomal acidification (N = 3 / group). (L and M) Representative images and quantification of lysosomal regions in transmission electron micrographs (N = 20 / group). Yellow arrows indicate lamellar inclusions, reflecting lipid accumulation within lysosomes. (N and O) IF staining of colocalization (yellow) of neutral sterols (BOPIDY, green) and acidic lysosomes (LysoTracker, red). Colocalization was measured using EzColocalization and plotted as Pearson correlation coefficients (N = 15 cells / group). Unless otherwise noted, all data were analyzed by two-way ANOVA and presented as mean ± SD.

[0098] Figure 7 .NCOA7 binding partner ATP6V1B2 is similarly upregulated in inflammatory models of PH. (A to H) ATP6V1B2 (red), CD31 (red), and CD41 (red) in pulmonary vessels of rodent PH models (N = 5-7 / group) and humans with PAH (N = 6-7 / group). + EC (green), α-SMA + Immunofluorescence staining and quantification of smooth muscle cells (white) and DAPI-stained nuclei (blue). All data were analyzed by Student's t-test and presented as mean ± SD.

[0099] Figures 8A-8B.NCOA7 deficiency reprograms sterol metabolism to upregulate oxysterols and bile acids. (A) Transcriptome analysis of PAECs under IL-1β by RNAi against control or NCOA7 (N=3 / group). Z-scores are indicated in blue for positive values ​​and in gold for negative values. FDR-corrected P-values ​​of identified cholesterol metabolism genes were <0.05. (B) Gene set enrichment analysis of the top 15 pathways by FDR-adjusted P-values, most of which were related to sterol metabolism and homeostasis (highlighted by red arrows). (C) Expression of LDLR detected by RT-qPCR (N=3 / group). (D and E) Flow cytometric analysis of fluorescently labeled NBD-cholesterol uptake (N=3 / group). (F) Expression of PAECs under siNC or siNCOA7, or (G) using lentiviral GFP, NCOA7 by relative luminescence measurement. 短 or NCOA7 全长 Overexpressed total cholesterol content. Data were analyzed by one-way ANOVA (N=6 / group). (H) Expression of CH25H detected by RT-qPCR (N=3 / group). (I to N) CH25H (red), CD31 in pulmonary vessels of mouse (N=8 / group), rat (N=5 / group) and human (N=6-7 / group) PH models. + EC (green), α-SMA + IF staining and quantification of smooth muscle cells (white) and DAPI-stained nuclei (blue). Student t-test. (O to Q) Targeted oxysterol quantification by LC-MS (N = 5 / group) and (R to W) unbiased bile acid metabolite quantification by LC-MS, organized in shaded boxes by suggested pathways (N = 4-6 / group). Metabolite 7HOCA is shown in inset (W). Unless otherwise indicated, all data were analyzed by two-way ANOVA and are presented as mean ± SD.

[0100] Fig. 9A and 9B NCOA7 regulation of oxysterol and bile acid metabolism is independent of sterol flux in de novo cholesterol synthesis. (A to N) Direct measurement of post-squalene intermediates by liquid chromatography-mass spectrometry (N = 5 / group). (O and P) CH25H (red), CD31 + EC (green), α-SMA + Immunofluorescence staining and quantification of smooth muscle cells (white) and DAPI-stained nuclei (blue) (N=5 / group; Student t-test) Unless otherwise stated, all data were analyzed by two-way ANOVA and are presented as mean ± SD.

[0101] Figures 10A-10C .NCOA7-CH25H axis drives lung endothelial immune activation. (A to F) RT-qPCR and immunoblotting assays of NCOA7 in (A and B) RNAi targeting NCOA7 and (C and D) lentiviral delivery of NCOA7. 短 or NCOA7 全长 , or (E and F) VCAM1 expression under RNAi for NCOA7 and CH25H (N=3 / group). Immune cell adhesion of leukocytes (G to I) or monocytes (J to L) to endothelial monolayers (N=6 / group). (M and N) Expression of VCAM1 in PAEC treated with ethanol control and 7HOCA (50 μM) for 24 hours detected by RT-qPCR and immunoblotting (N=3 / group; Student t-test). (O and P) Leukocyte and monocyte adhesion in 7HOCA-treated PAEC compared with ethanol control (N=6 / group; Student t-test). Unless otherwise stated, all data were analyzed by two-way ANOVA and expressed as mean ± standard deviation.

[0102] Figures 11A-11C NCOA7 regulates endothelial cell apoptosis and proliferation and oxysterols immunoactivate endothelium. (A to C) Apoptosis detected by caspase-3 / 7 activity (N=6 / group; two-way ANOVA). (D to E) Proliferation detected by BrdU incorporation (N=6 / group; two-way ANOVA). (G to O) VCAM1 expression detected by RT-qPCR (N=3 / group), and adhesion of leukocytes or monocytes to monolayers treated with ethanol with 25HC (25 μM) or triol (5 μM) or tetraol (50 μM) for 24 hours (N=6 / group). (P to S) Immunoblotting of VCAM1 in PAEC under different conditions, repeated three times (red boxes indicate presentation in the main figure). Unless otherwise stated, all data were analyzed by Student's t-test and are presented as mean ± standard deviation.

[0103] Fig. 12A and 12B.The G allele of SNP rs11154337 prevents lysosomal lipid accumulation and attenuates oxysterol-mediated immune activation in iPSC-ECs. (A to C) Clinical readout and survival of SNP rs11154337 allele variants and their 6MWD in PAH patients in the UPMC cohort (N=93) and STRIDE cohort (N=630). (D) Schematic diagram of iPSC-EC generation. (E and F) Expression of NCOA7 isoforms detected by RT-qPCR (N=3 / group). (G and H) SiR-lysosomal dye for active cathepsin D (purple; N=10 cells / group). (I) ATP6V1B2 expression detected by RT-qPCR (N=3 / group). (J) Transmission electron micrograph quantification of lysosomal area. (K) Total cholesterol content measured by relative luminescence (N=6 / group). (L and M) BODIPY dye for neutral lipids (green; N = 10 cells / group). (N) CH25H expression detected by RT-qPCR (N = 3 / group). (O) Targeted 25HC quantification by LC-MS (N = 5 / group). (P and Q) VCAM1 expression detected by RT-qPCR or immunoblotting (N = 3 / group). (R and S) Adhesion of leukocytes and monocytes to iPSC-EC monolayers (N = 6 / group). Unless otherwise stated, all data were analyzed by two-way ANOVA and are presented as mean ± SD.

[0104] Fig.13 Genetic deletion of Ncoa7 does not alter left ventricular function but upregulates plasma oxysterols and bile acids. (A to E) (A to E) Ncoa7 - / - x + Echocardiographic measurements of heart rate, left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and left ventricular posterior wall distance during diastole and systole (LVPW; d and LVPW; s) in mice (N = 3-5 / group) and (J to N) PBS or 7HOCA (10 mg / kg) mice under four weeks of hypoxia (N = 3-6 / group). (F to I) Il6Tg measured using LC-MS + With Ncoa7 - / - x + 7HOCA in mouse serum (N=5-8 / group) Unless otherwise stated, all data were analyzed by Student's t-test and expressed as mean±SD.

[0105] Fig.14Genetic deletion of NCOA7 and oral tracheal delivery of 7HOCA exacerbate PAH in vivo. (A) Ncoa7-null mice were crossed with the Il6 Tg+ PAH model. (B to F) Target protein staining (i.e., CH25H, VCAM1, or CD11b; red), endothelial layer (CD31; green), smooth muscle layer (α-SMA; white), and nuclear counterstaining (DAPI; blue) of pulmonary vessels of Il6 Tg+ and Ncoa7- / -x Il6 Tg+ mice were performed. The relative intensity of CH25H or VCAM1, the number of CD11b+ cells per vessel, or the degree of vascular muscularization defined by the thickness of the α-SMA layer and the total vessel diameter were quantified. N = 8 / group. (G) 7HOCA was measured in the serum of Il6 Tg+ and Ncoa7- / -x Il6Tg+ mice using LC-MS. N = 5-8 / group. (H) Fulton index and (I) RVSP of Il6 Tg+ and Ncoa7- / -x Il6Tg+ mice. N=6-10 / group. (J) Mice received orotracheal delivery of PBS or 7HOCA (10 mg / kg) under hypoxic (10% O2) conditions for four weeks and were sacrificed at 16 weeks. (K to N) Target protein staining of pulmonary vessels (i.e., VCAM1 or CD11b; red), endothelial layer (CD31; green), smooth muscle layer (α-SMA; white), and nuclear counterstaining (DAPI; blue) of PBS and 7HOCA mice. Quantification of relative intensity of VCAM, number of CD11b+ cells per vessel, or degree of vascular muscularization defined by α-SMA layer thickness and total vessel diameter. N=4 / group. (O) Fulton index and (P) RVSP of mice receiving orotracheal PBS or 7HOCA. N=4-7 / group. Unless otherwise stated, all data were analyzed by Student's t-test and presented as mean ± SD.

[0106] Fig.15A and 15B.Genomic structure of NCOA7 and generation of SNP-edited iPSC-derived ECs. (A) High-throughput chromatin conformation capture was performed on human umbilical vein endothelial cells (GEO IDs GSM3438650 and GSM3438651). Blue columns represent the chromatin interaction frequencies with debiased, and purple dots represent the distance-normalized interaction frequencies. The arcs represented in indigo represent the interactions of the identified SNP rs11154337 (red), which are defined by the green threshold line. Data are obtained from the 3D Genomic Interaction Viewer and Database (3DIV; 3div.kr). The genetic maps of NCOA7 subtypes are shown below. (B) 3C assay in human PAECs predicts the interaction between the 3' end of a restriction enzyme (BspHI) digested genomic DNA fragment containing the NCOA7 promoter (N3) and the 5' end of a genomic DNA fragment containing SNP rs11154337 (S5), which generates a fusion sequence ligated at the BspHI cleavage site (N3S5). DNA gels confirm the presence of a 107 bp PCR product with primers targeting the fusion sequence spanning the BspHI cleavage site. Unligated genomic DNA was used as a PCR control. (C) DNA sequencing of PCR products to confirm the N3S5 fusion sequence. (D) ChIP-qPCR for p65 / RelA binding to the SNP rs11154337 region (N=3 / group). (E) Schematic representation of the cohorts used. (F) DNA sequencing of SNP rs11154337 in CRISPR-Cas9, SNP-edited iPSCs. (G) Flow cytometric analysis of sorting efficiency of iPSC-derived ECs by MACS. (H) Observation of iPSC-EC morphology by bright field, functional capacity by vascularization, and immunofluorescence staining of EC-specific markers CD144 (green) and CD31 (red). (I) Transmission electron microscopy of iPSC-EC. (J) Immunoblotting of VCAM1 in iPSC-EC, repeated three times (red box indicates presentation in the main figure). Unless otherwise stated, all data were analyzed by Student's t-test and presented as mean ± SD.

[0107] Figures 16A-16C .Computational Modeling Identification 958 ami A novel NCOA7 activator that abolishes endothelial immune activation and PAH. (A to C) The computational protocol for identifying small molecule modulators of NCOA7, consisting of three main parts: (A) druggability simulation, (B) pharmacophore modeling, and (C) virtual screening. (D and E) After MD simulation, the identified compound 958 was refined into its analog 958 ami. The interactions of compound atoms with NCOA7 residues are shown in two dimensions. Stronger interactions are shown in orange dashed lines, while weaker interactions are shown in gray dashed lines. The solid orange lines are strong / persistent interactions, with cumulative durations of more than 0.3 μs per interaction in the 0.6 μs total simulation. (F and G) Binding of the V-ATPase subunit ATP6V1B2 to NCOA7 (orange) was measured by proximity ligation assay. The insets show the binding of ATP6V1B2 and NCOA7 antibodies with DMSO or 958 ami Double incubation of 958 cells. Quantification of amplified signals per cell (N = 15 cells / group). (H) CH25H expression detected by RT-qPCR (N = 3 / group; two-way ANOVA). (I and J) DMSO compared to 958 cells detected by RT-qPCR and immunoblotting. ami VCAM1 expression in PAEC treated with DMSO (20 μM) for 24 hours (N=3 / group; two-way ANOVA). (K and L) Compared with DMSO control, 958 ami The adhesion rate of leukocytes and monocytes in PAEC treated with 958 (N = 6 / group). ami (7.5 mg / kg ip) one week before, rats were loaded with monocrotaline (80 mg / kg ip) for 10 days. (N to R) DMSO and 958 ami Target protein staining of pulmonary vessels (i.e., CH25H, VCAM1, or CD11b; red), endothelium (CD31; green), smooth muscle layer (α-SMA; white), and nuclear counterstaining (DAPI; blue) of rats. Quantification of the relative intensity of CH25H or VCAM1, CD11b in each vessel + Cell number or degree of vascular muscularization defined by α-SMA layer thickness and total vessel diameter. N = 6-7 / group. ami (S) Fulton index and (T) RVSP of monocrotaline-treated rats. N = 5-15 / group. Unless otherwise stated, all data were analyzed by Student's t-test and expressed as mean ± SD.

[0108] Fig.17Gaussian network model analysis of NCOA7 structural dynamics. (A) Mobility characteristics of residues in the three most collective motion modes of the NCOA7 catalytic domain. The main hinge residues for each mode are indicated by arrows and labeled. (B) For the three most cooperative modes, color-coded structures with regions exhibiting the greatest conformational flexibility are indicated in red, and regions with the least flexibility are indicated in blue. The hinge residues for each mode are shown as spheres and labeled.

[0109] Fig.18 Compounds 958 and 958 observed in molecular dynamics simulations ami Contact duration for binding to NCOA7. (A) Contact duration for NCOA7 residues from MD simulations (three independent runs, 0.2 ms each; 0.6 μs total for each compound). 958 is shown as cyan filled squares, and 958 ami Shown as magenta filled squares. Contact is defined as the cutoff distance between any heavy atom of the residue and the compound. (B) 958 and 958 ami 2D structure and atom ID numbers of NCOA7. (C) Nine graphs showing the duration of contacts between specific residues of NCOA7 (listed on the X-axis in A) and atoms of the compounds.

[0110] Fig.19 .NCOA7 observed in MD simulations with 958 and 958 ami Composite binding affinities. (A) The left panel shows the evolution of the binding affinity of the two compounds over time, referring to three independent runs for each system. The right histogram was obtained by compiling snapshots of all three runs for each compound. The average binding affinity (in kcal / mol) and the corresponding standard deviation (over the entire run) are indicated in each case. (B) At 200 ns, 958 ami The binding pose.

[0111] Fig. 20 .958 ami It did not change the left ventricular function of rats, nor did it cause hepatotoxicity or nephrotoxicity. (A) Treatment with DMSO or 958 ami Immunoblotting of VCAM1 in human PAEC treated with DMSO or 958 amiEchocardiographic measurements of heart rate, left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and left ventricular posterior wall distance during diastole and systole (LVPW; d and LVPW; s) in monocrotaline rats treated with (7.5 mg / kg ip) (N = 3-4 / group). (G and H) Heart rate and mean arterial pressure (MAP) during right heart catheterization (N = 6-15 / group). (I to K) DMSO or 958 ami Expression of Gpt and Got1 in liver tissue and Cst3 in kidney tissue of treated rats (N=5-6 / group) Unless otherwise indicated, all data were analyzed by Student's t-test and expressed as mean±SD.

[0112] Fig.21 .SNPs rs11154337 and 958 regulate entry of various pseudotyped enveloped viruses. (A to D) iPSCs transfected with human ACE2 receptor show reduced entry of multiple pseudotyped coronaviruses and herpes viruses in the presence of the G allele at SNP rs11154337, which leads to increased NCOA7 expression. (E) Application of the NCOA7 activator 958 enhances NCOA7 activity in HEK293 cells transfected with human ACE2 receptor to prevent infection with pseudotyped SARS-CoV-2 (D614G spike). (F and G) 958 parent (2 μM) and 958 ami (5 μM) reduced BA.2 spike pseudotyped virus infection in iPSCs transfected with human ACE2 receptor. (H and I) 958 parent (2 μM) and 958 ami (5 μM) reduces D614G spike pseudotype virus infection in iPSCs transfected with human ACE2 receptor. Unless otherwise stated, all data were analyzed by Student's t-test and presented as mean ± SD.

[0113] Fig. 22 . Reduced lung inflammation and mortality in coronavirus-infected human ACE2 transgenic mice treated with the NCOA7 activator 958. (A and B) Human ACE2 transgenic mice infected with two different coronavirus strains showed significantly improved mortality when treated with 958. (C to H) Lung tissues of mice treated with 958 showed reduced viral load and attenuation of various proinflammatory markers (IL-1α, IL-1β, IFN-γ, VCAM1, and ICAM1) as detected by RT-qPCR. Unless otherwise stated, all data were analyzed by Student's t-test and are presented as mean ± SD.

[0114] Fig.23.Mice infected with Klebsiella pneumoniae exhibited improved acute lung injury when treated with the NCOA7 activator 958. (A and B) Representative images of lung tissue sections stained with H&E from mice treated with DMSOComp.958 48 hours after intratracheal infection with Klebsiella pneumoniae. Black arrows represent neutrophils, while red arrows represent alveolar edema (400× original magnification, 50 μm scale bar). Acute lung injury was reduced in mice treated with 958. (C to E) Lung homogenate ELISA of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6. Unless otherwise stated, all data were analyzed by Student's t-test and are presented as mean ± SD.

[0115] Fig.24 .NCOA7 deficiency worsens survival in an ischemic stroke mouse model. A. Wild-type mice and NCOA7-deficient mice were used in an experimental model of ischemic stroke. Mice received sham surgery or transient middle cerebral artery occlusion (tMCAO) for 60 min, followed by restoration of cerebral blood flow. Then, mice were collected at 24 and 48 h for further studies. B. NCOA7 knockout mice had significantly reduced survival at 24 and 48 h after tMCAO compared with wild-type controls. There was no significant change in body weight at 24 or 48 h after tMCAO. C. Regional cerebral blood flow (rCBF) in the contralateral (CL) and ipsilateral (IL) cerebral cortex of mice was quantified using laser Doppler flowmetry. NCOA7-deficient mice had increased rCBF in the IL cerebral cortex at 24 h after tMCAO compared with wild-type controls. All data were analyzed as Student's t-test (*P<0.05, **P<0.01) and are presented as mean + / - SD.

[0116] Fig.25 .NCOA7 deficiency increases infarct volume and capillary leakage after ischemic stroke in mice. A. Mouse brain tissue was cut into 30-micron sections before immunofluorescence staining. Brain sections were stained with the neuronal marker microtubule-associated protein 2 (MAP2, green) to assess infarct volume after tMCAO. NCOA7-deficient mice had significantly increased infarct volume after ischemic stroke, indicating more neuronal cell death. There was no significant change in tissue swelling compared with wild-type controls. B. To assess capillary leakage, brain sections were stained for the plasma protein albumin (red). Notably, NCOA7-deficient mice had more albumin leakage into the brain tissue after tMCAO, indicating more severe brain microvascular damage compared with wild-type controls. All data were analyzed as Student's t-test (*P<0.05, **P<0.01) and are presented as mean + / - standard deviation.

[0117] Fig.26.NCOA7 deficiency leads to worsened neuroinflammation after ischemic stroke. A. Mouse brain sections were stained using an immunofluorescence protocol. The brain was stained with the astrocyte marker glial fibrillary acidic protein (GFAP, green), the microglial marker ionized calcium binding adapter molecule 1 (IBA1, red), and the nuclear marker (DAPI, blue). Images of the cortex (Ctx) and striatum (Str) of wild-type and NCOA7 knockout mice after tMCAO were acquired using confocal microscopy. Neuroinflammation in NCOA7-deficient mice was significantly worsened, as evidenced by swelling of astrocyte processes (green) in cortical and striatal tissues. In addition, microglia (red) in knockout mice were hypertrophic and elongated compared with wild-type controls. These data indicate that there is a large amount of reactive gliosis and neuroinflammation in the NCOA7-deficient brain after tMCAO.

[0118] Fig. 27 .In an ischemic stroke mouse model, NCOA7 deficiency leads to significant hypermyelination. A. Brain sections were stained for myelin basic protein (MBP, green) using an immunofluorescence protocol. Myelination in NCOA7-deficient mice was significantly enhanced overall, especially at the level of the corpus callosum (CC) and external capsule (EC). Notably, there was no significant difference in the contralateral (CL) hemisphere compared with the ipsilateral (IL) hemisphere after tMCAO. All data were analyzed as Student's t-test (*P<0.05, **P<0.01) and are expressed as mean + / - standard deviation.

[0119] Figures 28A-28E Exemplary structures of various compounds described herein are provided. DETAILED DESCRIPTION

[0120] The following description is merely exemplary in nature and is not intended to limit the present invention, its application or use. Although this description is intended to allow one of ordinary skill in the art to prepare and use the present invention, and specific examples are provided for this purpose, they should not be considered as limiting in any way. It will be apparent to one of ordinary skill in the art that various modifications to the following will fall within the scope of the appended claims. The present invention should not be considered limited to the aspects currently disclosed, whether in the examples or provided elsewhere herein.

[0121] The use of numerical values ​​within the various ranges specified in this application, unless otherwise expressly stated, is expressed as approximate values, as if the minimum and maximum values ​​within the range were preceded by the term "about". In this way, slight changes above or below the range can be used to achieve substantially the same results as the values ​​within the range. In addition, unless otherwise stated, the disclosure of these ranges is intended to be a continuous range including each value between the minimum and maximum values. For the definitions provided herein, these definitions refer to the word forms, cognates, and grammatical variants of these words or phrases. "A and an" used herein refers to one or more. The patent publications cited below are incorporated herein by reference in their entirety insofar as their technical disclosures and in accordance with this specification are consistent.

[0122] As used herein, the term "comprising, comprise or comprised" and its variants are open ended and do not exclude the presence of unidentified additional elements. In contrast, the term "consisting of" and its variants are intended to be closed ended and exclude any additional elements except trace amounts.

[0123] As used herein, the term "patient" or "subject" refers to members of the animal kingdom, including, but not limited to, humans, and "mammal" refers to all mammals, including, but not limited to, humans.

[0124] As used herein, "treatment" or "treating" inflammation, cardiovascular inflammation, vascular inflammation (e.g., endothelial inflammation), diseases characterized by vascular inflammation, or innate and acquired immune diseases means administering a composition, device or structure to a patient by any suitable dosing regimen, procedure and / or route of administration to achieve a desired clinical / medical endpoint, including, but not limited to, for PAH, a mean pulmonary artery pressure of ≥ 25 mmHg as measured by resting supine right heart catheterization. Reducing or preventing further development of vascular endothelial inflammation (e.g., PAH). The amount of any agent or therapeutic agent administered by any suitable route that is effective in treating a patient is an amount that is capable of preventing, reducing and / or eliminating endothelial inflammation (e.g., PAH) and / or reducing the severity of one or more symptoms of endothelial inflammation (e.g., PAH), for example, a mean pulmonary artery pressure of < 25 mmHg as measured by resting supine right heart catheterization. The therapeutically effective amount of each therapeutic agent can range from 1 pg per dose to 10 g per dose, including any amount therebetween, such as, but not limited to, 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, and, for example, as a single dose or bolus, at regular or irregular intervals, in an amount and interval dictated by any clinical parameter of the patient, or continuously.

[0125] Cardiovascular inflammation, such as vascular inflammation (e.g., endothelial inflammation or vascular endothelial inflammation), may be associated not only with PAH, but also with other diseases, including, but not limited to: peripheral arterial disease, vasculitis (including large, medium and small vessel inflammation), infectious diseases, chronic vascular inflammatory diseases (e.g., atherosclerosis), inflammatory or inflammation-related diseases (e.g., restenosis), essential hypertension and stroke (see, e.g., 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 (e.g., heart failure (HFpEF and HFrEF)), myocarditis and atrial fibrillation, in which inflammatory myeloid cells also appear to worsen the symptoms and severity of the disease. Pulmonary hypertension, including PAH, pulmonary hypertension caused by left heart disease, pulmonary hypertension caused by lung disease, pulmonary hypertension caused by chronic thrombosis in the lungs, and pulmonary hypertension of unknown etiology, can be effectively treated by the compositions and methods described herein. Other innate and acquired immune diseases include, for example, heart disease, lung disease, sepsis, cancer, and neurodegenerative diseases, for example, inflammation associated with, for example, heart disease, lung disease, sepsis, cancer, and neurodegenerative diseases.

[0126] As used herein, "treatment" or "treating" coronavirus infection means administering a composition, device or structure to a patient by any suitable dosing regimen, procedure and / or route of administration to achieve a desired clinical / medical endpoint, including, but not limited to, for coronavirus infection, reducing or preventing further development of coronavirus infection, for example, as determined below. The amount of any agent or therapeutic agent administered by any suitable route to effectively treat a patient is an amount that can prevent, reduce and / or eliminate coronavirus infection and / or reduce the severity of one or more symptoms of 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, stuffy or runny nose, nausea or vomiting or diarrhea. Each therapeutic agent (e.g., compound (1) described below) is 958 and 958 ami The therapeutically effective amount (for example) can range from 1 pg per dose to 10 g per dose, including any amount therebetween, such as, but not limited to, 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, and for example, as a single dose or a large dose, at regular or irregular intervals, in an amount and interval dictated by any clinical parameter of the patient, or continuously.

[0127] Active ingredients (e.g., compound (1) described below, 958 and 958 ami For example, the compound may be compounded or otherwise made into a composition suitable for use, such as a pharmaceutical dosage form or a pharmaceutical product with the compound as an active ingredient. The composition may include a pharmaceutically acceptable carrier or excipient. An excipient is an inactive substance used as a carrier of a pharmaceutically active ingredient. Although "inactive", an excipient can promote and help improve the delivery or bioavailability of the active ingredient in a drug. Non-limiting examples of useful excipients include: anti-adherents, adhesives, rheology modifiers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavoring agents, colorants, glidants, lubricants, preservatives, antioxidants, adsorbents, vitamins, sweeteners, etc., such as those available in the pharmaceutical / compounding field.

[0128] Useful dosage forms include, 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 the active ingredient (drug or compound) and a solvent (e.g., water, saline, lactated Ringer's solution, or phosphate buffered saline (PBS)). Other excipients, such as polyethylene glycol, emulsifiers, salts, and buffers, may be included in the solution.

[0129] Suitable dosage forms may include single-dose or multi-dose vials or other containers, such as a medical syringe or dropper, containing a composition comprising the active ingredients described herein for treating coronavirus infection.

[0130] Pharmaceutical preparations suitable for use include aqueous and non-aqueous sterile solutions, which, in addition to active pharmaceutical ingredients or drugs, may also contain, for example, but not limited to, antioxidants, buffers, antibacterial agents, lipids, liposomes, lipid nanoparticles, emulsifiers, suspending agents, and rheology modifiers. Preparations may be present in unit dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored under freeze drying (lyophilization) conditions, requiring only the addition of sterile liquid carriers, for example, water for injection, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0131] The therapeutic composition must usually be sterile and stable under production and storage conditions. For example, a sterile injectable solution can be prepared by incorporating the required amount of active agent with one or a combination of the ingredients listed herein (as required) into an appropriate solvent, followed by filtration sterilization. Typically, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, typical preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional required ingredients from its previous sterile filtered solution. The appropriate fluidity of the solution can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The absorption of the injectable composition can be extended by including an agent that delays absorption (e.g., monostearate and gelatin) in the injectable composition.

[0132] "Therapeutically effective amount" refers to the amount of a pharmaceutical product or active agent that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. An "effective amount" for treating a condition is the amount of an active agent or dosage form (e.g., single dose or multiple doses) that effectively reaches a determinable endpoint. "Effective amount" is preferably safe-at least to the extent that the therapeutic benefit is greater than the damage, and / or the damage is acceptable to ordinary technicians and / or appropriate regulatory agencies (e.g., the U.S. Food and Drug Administration). The therapeutically effective amount of an active agent can vary according to factors such as individual disease state, age, sex, and body weight, and the ability of the active agent to cause a desired response in an individual. "Preventive effective amount" refers to the amount that can effectively achieve the desired preventive outcome within the necessary dosage and time period. Typically, since the preventive dose is used in a subject before or in the early stages of the disease, the preventive effective amount may be lower than the therapeutically effective amount.

[0133] The dosage regimen can be adjusted to provide the best desired response (e.g., a therapeutic or preventive response). For example, a single dose or a large dose can be administered, multiple divided doses can be administered over a period of time, or the composition can be administered continuously or in a pulsed manner, wherein the dose or partial dose is administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90 or 120 minutes, every 2 to 12 hours per day, or every other day, etc., proportionally reduced or increased according to the urgency of the treatment situation. In some cases, it may be particularly advantageous to prepare the composition in a dosage unit form for ease of administration and uniform dosage. The specifications of the dosage unit form depend on and are directly dependent on (a) the unique properties of the active compound and the specific therapeutic or preventive effect to be achieved, and (b) the inherent limitations of the technology for preparing such active compounds for treating individual sensitivity.

[0134] An amount effective for treating inflammation or an inflammatory condition in a patient can be 1 μg / kg to 10 g / kg, or 1 ng / kg to 100 mg / kg of compound 958 per day, for example, an amount of the compound that produces 20 μM ± 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 0.5%, 1% (e.g., 1 to 40 μM) or any increment therebetween in the patient's body fluids (e.g., blood, serum, plasma, etc.).

[0135] Virus-related inflammation (e.g., respiratory inflammation) can also be treated using the compounds described herein. For example, coronaviruses are a group of related RNA viruses that cause disease in mammals and birds. In humans and birds, they cause respiratory infections ranging from mild to fatal. Mild illnesses in humans include some common colds (also caused by other viruses, mainly rhinoviruses), while more deadly varieties can cause Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) or 2019 Coronavirus Disease (COVID-19). In cattle and pigs, coronaviruses cause diarrhea, while in mice, they cause hepatitis and encephalomyelitis.

[0136] SARS-CoV-2 is the virus that causes COVID-19, the respiratory disease that is responsible for the COVID-19 pandemic. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that is contagious in humans. Each SARS-CoV-2 virion is about 50-200 nanometers in diameter. SARS-CoV-2 has four structural proteins, called S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein contains the RNA genome, and the S, E, and M proteins together build the viral envelope. The spike protein is the protein responsible for attaching the virus to and fusing with the host cell membrane; specifically, its S1 subunit catalyzes attachment and the S2 subunit catalyzes fusion. Symptoms of SARS-CoV-2 infection may appear within 2-14 days after exposure to the virus. Symptoms of SARS-CoV-2 may 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, stuffy or runny nose, nausea or vomiting, or diarrhea.

[0137] As shown below, it is proved that compounds 958 and 958 amiCan be used to treat inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with vascular endothelial inflammation as a symptom, such as arterial pulmonary 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 neurodegenerative diseases, for example, inflammation associated with heart disease, lung disease, sepsis, cancer and neurodegenerative diseases. The compound can be used to treat viral infections, such as inflammation associated with viral infections, such as SARS-CoV-2 or other SARS or MERS virus infections. Compound 958, such as 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-phenyl3,4-dihydrophthalazine-1-carboxylate, which can have the following structure:

[0138] and include pharmaceutically acceptable salts thereof, and may include equivalent derivative compounds, such as esters or prodrugs thereof.

[0139] Compound 958 ami Has the following structure:

[0140]

[0141] Compounds 958 and 958 ami is an example of compound (1) provided herein, which has the following general structure and is expected to have similar activities:

[0142]

[0143] Wherein R1 and R2 are both or individually (independently) -H or -C 1-3 alkyl;

[0144] Z is O or NH;

[0145] X1, X2, and X3 are independently N or C;

[0146] X4 is located at the ortho, meta or para position to X1 and is N or C;

[0147] Y2 is -H, -C 1-3 Alkyl, halogen (-F, -Cl, -Br or -I) or -NO2 (nitro);

[0148] Y1 is -H, -C 1-3 Alkyl, halogen (-F, -Cl, -Br or -I), -NO2, -CN (nitrile), -CF3 (trifluoromethyl), -SO2R4 (wherein R4 is -OH, -C 1-3 alkyl), -NHR5 (wherein R5 is H, -C 1-3 alkyl), -NHR6 (wherein R6 can be -H, -C 1-3 alkyl), -NHC(O)-R7 (wherein R7 can be -H, -C 1-3 alkyl), -OR8 (wherein R8 can be -H, -C 1-3 alkyl), -OC(O)-R9 (wherein R9 can be -H, -C 1-3 Alkyl or C 1-3 Alkoxy), -C(O)-R 10 (where R 10 Can be -H, -C 1-3 alkyl) or -C(O)-R 11 -R 12 (R 11 Can be O or NH; R 12 Can be -H, -C 1-3 alkyl), and includes pharmaceutically acceptable salts thereof, and may include equivalent derivative compounds, such as esters or prodrugs thereof. In the case where the "R group" is an ester or amide, the ester or amide may form a bond connecting the active moiety to another moiety (e.g., an inactive moiety or a carrier). As used herein, "moiety" refers to a portion of a molecule, such as a moiety that can be attributed to activity or functionality.

[0149] As used herein, "alkyl" refers to a straight chain, branched chain or cyclic hydrocarbon (hydrocarbyl) group, including, for example, 1 to about 24 carbon atoms, such as but not limited to C containing 1, 2 or 3 carbon atoms. 1-3 "Alkoxy" refers to an alkyl group attached through an oxygen, such as methoxy (-OCH3), ethoxy (-O-CH2-CH3) or propoxy (e.g., -O-CH2-CH2-CH3 or -O-CH(CH3)2), 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, including but not limited to ethylene (-CH2-CH2-).

[0150] Compound (1) can be used to treat a patient as described above with respect to compounds 958 and 958 amiThe invention relates to, for example, treating inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with vascular endothelial inflammation as a symptom, such as arterial pulmonary hypertension, restenosis, essential hypertension, atherosclerosis and stroke), diseases characterized by vascular inflammation, or innate and acquired immune diseases, such as autoimmune diseases, heart disease, lung disease, sepsis, cancer and neurodegenerative diseases, such as inflammation associated with heart disease, lung disease, sepsis, cancer and neurodegenerative diseases. The compound can be used to treat viral infections, such as inflammation associated with viral infections, such as SARS-CoV-2 or other SARS or MERS virus infections.

[0151] The therapeutically effective amount of each compound for treating a disease described herein can range from 1 pg per dose to 10 g per dose, including any amount therebetween, such as, but not limited to, administering to a patient 1 ng, 1 μg, 1 mg, 10 mg, 100 mg, or 1 g per dose, such as 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 can range from 1 μg / kg to 10 g / kg per day, or 1 ng / kg to 100 mg / kg of compound 958, or an equivalent amount of another compound described herein, or 20 μM ± 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, .5%, 1%, or any increments therebetween in a patient's body fluid (e.g., blood, serum, plasma, etc.). The therapeutic agent can 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 amounts and intervals dictated by any clinical parameter of the patient.

[0152] As shown below, additional compounds that can be used to treat the following diseases are evaluated: inflammation and inflammatory diseases, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with vascular endothelial inflammation as a symptom, such as arterial pulmonary 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 neurodegenerative diseases, such as inflammation associated with heart disease, lung disease, sepsis, cancer and neurodegenerative diseases. The compounds can be used to treat viral infections, such as inflammation associated with viral infections, such as SARS-CoV-2 or other SARS or MERS viral infections. The compounds listed in Figures 11, 16A-16E can be provided as pharmaceutically acceptable salts thereof, and can include equivalent derivative compounds, such as esters or prodrugs thereof.

[0153] Single nucleotide polymorphism (SNP) rs11154337 is a polymorphism within the NCOA7 gene. Figure 1 and 2 The sequence and position of rs11154337 in an intron of the NCOA7 gene are shown (see also, U.S. Patent Publication No. 2021 / 0309998A1, the disclosure of which is incorporated herein by reference in its entirety). Figure 3A and 3B Exemplary NCOA7 mRNA (cDNA) sequences are provided.

[0154] NCOA7 is the protein product of the NCOA7 gene. Figure 3A and 3B An exemplary NCOA7 mRNA sequence is provided (SEQ ID NO: 2). The NCOA7 gene has a candidate SNP, designated rs11154337 (SEQ ID NO: 1). Figure 1 and 2 An exemplary rs11154337 sequence is provided. Rs11154337 is located in the promoter of the interferon-induced NCOA7 isoform (NCOA7 short), which was first discovered in our unpublished genome-wide association study (GWAS) of survival in human pulmonary hypertension. Other mutations or polymorphisms located in the same intron as rs11154337 or in linkage disequilibrium with rs11154337 (e.g., Figure 2rs11154337), may indicate the presence of a high-risk genotype and may functionally affect the expression of NCOA7 and may therefore, like or in combination with rs11154337, help detect people who are particularly susceptible to coronavirus and correct risk polymorphisms, such as functional polymorphisms affecting NCOA7 expression through gene editing, may reduce coronavirus infectivity. SNPrs11154337 (SEQ ID NO:1) is present in an intronic region where both the RelA / p65 subunit of NF-kB and STAT1 are expected to bind. From an antimicrobial defense perspective, this duality suggests a functional collaboration between two host defense pathways: (1) initial detection at the plasma membrane through Toll-like receptors and the NF-kB pathway and (2) potential endosomal pathogen escape, which triggers an interferon-mediated response and activation of STAT1 / 2 through Janus tyrosine kinases (JAKs). Thus, our molecular studies have defined NCOA7 as an upregulated factor in EC in response to proinflammatory cytokines; moreover, inhibition of STAT1 / 2 signaling by the JAK inhibitor momelotinib and RNAi of RelA / p65 both abrogated IL-1β-mediated NCOA7 upregulation. We have demonstrated that NCOA7 regulates immune activation of the endothelium and subsequent leukocyte adhesion and possible infiltration. To this end, NCOA7 alters lysosomal acidification, a property that has been independently found to affect the entry of other enveloped viruses, such as influenza virus. Furthermore, using in vitro biochemical analysis of nucleoprotein binding to SNP rs11154337 and CRISPR-Cas9-edited isogenic induced pluripotent stem cells (iPSCs), we found that allele-specific binding to the NF-kB subunit RelA / p65 drives allele-specific expression of NCOA7.

[0155] Lysosomes are membrane-bound organelles found in many animal cells. Lysosomes are spherical vesicles that contain hydrolases that can break down a variety of biomolecules. Lysosomes have a specific composition of their membrane proteins and their luminal proteins. The pH of the lumen (~4.5-5.0) is optimal for enzymes involved in hydrolysis, similar to the activity of the stomach. In addition to degrading polymers, lysosomes are involved in various cellular processes, including secretion, plasma membrane repair, apoptosis, cell signaling, and energy metabolism. Lysosomes act as a waste disposal system for cells by digesting used materials in the cytoplasm from inside and outside the cell. Materials from outside the cell are taken up by endocytosis, while materials 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 ranges from ~4.5-5.0, so the interior of lysosomes is acidic compared to the slightly alkaline cytosol (pH 7.2). Lysosomes transport protons (H +Ions) are pumped from the cytosol across the membrane into the lysosome, thus maintaining the pH difference. The vacuolar ATPase is responsible for the transport of protons, while the reverse transport of chloride ions is carried out by ClC-7Cl - / H + Reverse transporter (antiporter) carried out.

[0156] "Our work establishes a paradigm linking lysosomal biology and oxysterol and bile acid metabolism to endothelial inflammation, with broad implications for precision medicine approaches to arterial pulmonary hypertension. We also found evidence of genetic association of this NCOA7 SNP with essential hypertension, atherosclerosis, and stroke, making this target relevant to these other vascular diseases. In pilot data, we have found associations between these same oxysterols and bile acids in sepsis, suggesting that targeting NCOA7 will be beneficial in this critical illness as well as acquired and innate immune diseases in general."

[0157] In this process, we used computational prediction methods to identify specific small molecules that can activate or inhibit NCOA7 based on high predicted likelihood of binding to the enzymatic pocket, lysin motif (LysM), and catalytic (TLDc) domain of Tre2 / Bub2 / Cdc16 (TBC) of NCOA7. One of the top hit compounds, 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 activities of altering lysosomal pH and downregulating cholesterol metabolism as well as inflammatory activation of endothelial cells. They may also have such therapeutic activities for other non-vascular diseases of acquired and innate immunity. Finally, they may serve as novel therapeutics for COVID-19, which is supported by our experimental data showing that this compound can inhibit the entry of SARS-CoV-2 pseudotyped viruses into human cells.

[0158] In various aspects and embodiments:

[0159] 1. The invention is based on novel and unpublished mechanistic data describing the role of NCOA7 in controlling lysosomal activity, sterol homeostasis and inflammation.

[0160] 2. Notably, the present invention targets new pathways and a set of targets that have not been previously attempted in cardiovascular and pulmonary vascular diseases, sepsis or COVID-19.

[0161] 3. Importantly, the association of NCOA7 and target pathways with human arterial pulmonary hypertension, essential hypertension, stroke, and atherosclerosis is based on population-level human genetic and metabolomics data.

[0162] 4. (6,7-Dihydroxy-2-oxo-2H-chromen-4-yl)methyl 4-oxo-3-phenyl 3,4-dihydrophthalazine-1-carboxylate The molecule has no known protein targets and has not been studied in the context of any human disease or inflammation in general.

[0163] 5. The computational process used to make these small molecule predictions has never been applied to NCOA7 or the TLDc domain in the past.

[0164] In a further embodiment, a method of treating a patient having a C at SNP rs11154337 and having inflammation and / or an inflammatory disease (e.g., cardiovascular inflammation, vascular inflammation (e.g., diseases having vascular endothelial inflammation or having vascular endothelial inflammation as a symptom, such as arteriopulmonary hypertension, restenosis, essential hypertension, atherosclerosis, and stroke)), a disease characterized by vascular inflammation, or an innate and acquired immune disease, such as an autoimmune disease, heart disease, lung disease, sepsis, cancer, and neurodegenerative disease, e.g., inflammation associated with, e.g., heart disease, lung disease, sepsis, cancer, and neurodegenerative disease, is provided. The method includes using gene editing, such as but not limited to CRISPR / Cas9 or TALEN-based gene editing methods to change one or more Cs at SNP rs11154337 to Gs (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, which describes CRISPR / CAS9 technology and tools that can be used to implement such technology and tools, as well as commercial services for gene editing, such as using technology and expertise provided by CRISPR Therapeutics (Cambridge, MA).

[0165] Example

[0166] By utilizing large-scale, multidimensional metabolomics and genomics data and in vitro and in vivo mechanistic experiments, we found that NCOA7 controls lysosomal activity and endothelial steroid homeostasis, acting as a homeostatic brake, thereby moderating inflammation, endothelial dysfunction, and PAH caused by oxysterols and 7HOCA. In addition, when the G allele of the NCOA7 intronic SNP rs11154337 is present, NCOA7 is increased, thereby reducing inflammation in PAH, and providing mechanistic evidence for the genetic association of SNP rs11154337 with PAH disease severity, the metabolomic association of oxysterol and bile acid profiles with PAH severity, and the genetic association of SNP rs11154337 with 7HOCA plasma levels. Identification of active pharmaceutical ingredients (e.g., drugs) that can reduce inflammation (e.g., endothelial inflammation) provides useful therapeutic methods for treating various diseases associated with inflammation, including cardiovascular inflammation, vascular inflammation (e.g., diseases with or 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 used to illustrate and demonstrate the concepts of the claimed invention.

[0167] Example 1

[0168] Vascular inflammation critically regulates the pathological phenotype of endothelial cells (ECs), but the pathogenic mechanisms remain incompletely defined, particularly in pulmonary arterial hypertension (PAH). Immune dysregulation and metabolic reprogramming are well-established principles of PAH pathogenesis, but a unifying theory linking the two has yet to be established. In human pulmonary artery ECs, induction of nuclear receptor coactivator 7 (NCOA7), a gene previously identified as upregulated in PAH, moderates proinflammatory sterol production by enhancing lysosomal acidification and limiting EC immune activation. Conversely, reduced NCOA7 promotes lysosomal dysfunction, generating proinflammatory sterols and bile acids that drive an EC phenotype consistent with PAH. In vivo, NCOA7-deficient mice or mice treated with exogenous 7α-hydroxy-3-oxo-4-cholenic acid (7HOCA, a representative metabolite of the NCOA7-dependent sterol signature) exhibit exacerbated PAH. Unbiased, metabolome-wide association studies from a multicenter PAH biobank cohort (N = 2,796) highlighted the clinical importance of this mechanism in controlling disease severity, identifying plasma signatures associated with mortality in PAH, including the same NCOA7-dependent sterols and bile acids (adjusted P < 1.1 × 10 -6). 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, SNP-edited, and iPSC-derived ECs, suggesting that there may be a broad genetic susceptibility to NCOA7 deficiency. Correspondingly, in an independent, single-center PAH cohort (N=93), SNP rs11154337 was associated with PAH severity, as shown by six-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]). In a second validation multicenter 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 NCOA7 functional domains, we predicted and synthesized novel NCOA7 activators that prevented endothelial immune activation and reversed PAH markers in monocrotaline rats. In summary, this work establishes a genetic and metabolic paradigm linking lysosomal biology and sterol and bile acid processing to EC inflammation and suggests novel therapeutics. This paradigm has broad implications not only for the molecular diagnosis and therapeutic treatment of PAH, but also for other vascular diseases that rely on acquired and innate immune regulation.

[0169] Vascular inflammation critically regulates endothelial cell (EC) behavior in vascular diseases such as atherosclerosis, hypertension, stroke, and sepsis. In the lung, endothelial inflammation is a prominent feature of acute lung injury, pathogen-mediated processes such as SARS-CoV-2 infection, and pulmonary arterial hypertension (PAH), a fatal and mysterious vascular disease characterized by complex vascular remodeling and unclear molecular origins. However, the precise pathogenic role of inflammation in PAH has been controversial, and it is possible that inflammation controls PAH severity more clearly than overall risk.

[0170] Molecular homeostatic accelerators and brakes that regulate inflammation are essential for maintaining cellular function. However, the specific levers that control inflammation leading to EC dysfunction, such as in PAH, have not been fully described. Lysosomal activity is increasingly being viewed as a master regulator of inflammation, and dysfunctional lysosomal activity has been observed in PAH. Central to maintaining lysosomal enzyme function is proper acidification of the luminal space, which is controlled by the vacuolar H +The mitochondrial endothelial growth factor (NGF) is a family of ATPases (V-ATPases), and loss of this hydrolytic capacity results in diseases known as lysosomal storage diseases (LSDs), which have been reported to have a pulmonary vascular phenotype. Nuclear receptor coactivator 7 (NCOA7) directly binds to and regulates V-ATPase activity to control endolysosomal function, which has been shown to play a role in controlling bacterial and viral pathogen entry, tubular acidification, and neuronal function. NCOA7 is upregulated in human EC and PAH lung tissue by proinflammatory stimuli, but any pathogenic mechanisms linking NCOA7 to cardiopulmonary vascular disease have not been identified.

[0171] Downstream of acidification, lysosomes carry pH-sensitive hydrolases responsible for the breakdown of cellular waste products and macromolecular transport. Lysosomal-mediated breakdown of cellular waste products is associated with autophagy, a process that may be relevant to PAH. Notably, 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 patients with PAH. Oxysterols and bile acids influence cholesterol biosynthesis and cell membrane properties, driving key cellular defenses in adaptive and innate immunity. At the endothelial level, these molecules enable endothelial immune activation, thereby promoting peripheral vascular diseases such as atherosclerosis and hypertension. A recent unbiased plasma metabolomic analysis of 2,796 patients with PAH also found metabolome-wide associations between the profile of glucuronidated oxysterols and downstream bile acids and indicators of PAH disease severity and mortality (adjusted P < 1.1 × 10 -6 Therefore, given the intrinsic connection of NCOA7 to lysosomal biology and the metabolome-wide association of lysosomal-derived oxysterol and bile acid levels with PAH mortality, we sought to determine whether NCOA7 controls oxysterol and bile acid metabolism, inflammatory lung EC pathological phenotypes, and the development of PAH. Elucidating these links would provide a mechanistic explanation for the associations between glucuronidated oxysterols and bile acids and PAH mortality and severity.

[0172] method

[0173] RNA extraction and quantitative polymerase chain reaction: Cells were lysed in QIAzol lysis reagent (Qiagen; 79306), and RNA was extracted using the Rnaeasy kit (Qiagen; 74004). Complementary DNA was synthesized using a high-capacity cDNA reverse transcription kit (ThermoFisher; 4368813). Quantitative real-time PCR (RT-qPCR) was performed on an Applied Biosystems QuantStudio 6Flex real-time PCR instrument. Target gene expression was normalized to a housekeeping gene (i.e., ACTB) and 2 -ΔΔCt Fold changes were calculated by TaqMan TMUniversal PCR Master Mix (ThermoFisher; 4305719) was used with TaqMan primers (Table 1). TM SYBR TM Green Master Mix (ThermoFisher; A25742) was used together with custom designed primers (Table 2).

[0174] Table 1. TaqMan primers.

[0175] Gene Targets Species TaqMan Assay ID ACTB people Hs99999903_m1 ATP6V1B2 people Hs00156037_m1 CH25H people Hs02379634_s1 LDLR people Hs01092524_m1 NCOA7 people Hs00906291_m1 VCAM1 people Hs01003372_m1

[0176] Table 2. Custom primers used for gene expression.

[0177]

[0178] Immunofluorescence staining of lung tissue: OCT-embedded lung tissue was cut into thin slices of 5 to 7 microns thickness using a cryostat and subsequently fixed to gelatin-coated histological slides. The slices were rehydrated with PBS for five minutes, fixed in 2% PFA for 30 minutes, permeabilized in 0.1% Triton X-100 for 15 minutes, and blocked in 5% donkey serum and 2% BSA in PBS for one hour at room temperature. The primary antibody (Table 3) was diluted in 2% BSA and incubated overnight at 4°C. Alexa Fluor-conjugated secondary antibodies (ThermoFisher) were used at a dilution of 1:1000 in 2% BSA for one hour at room temperature. The slices were then counterstained with Hoescht for one minute at room temperature and then sealed. Small pulmonary vessels (30 to 100 microns in diameter) not associated with the bronchial airways were selected for imaging.

[0179] Table 3. Antibodies used for protein detection.

[0180]

[0181] Single-cell transcriptomics: Single-cell RNA sequencing was performed on the lungs of healthy controls and idiopathic PAH patients. Expression matrices were derived using CellRanger. Subsequent batch correction, scaling, and normalization were performed using SCTransform in Seurat v3. Cell types were determined with SingleR using the Blueprint ENCODE reference. Cells were identified as positive for NCOA7 if the transformed expression value was greater than 0. Cells expressing NCOA7 were identified as having a transformed expression value greater than 0.2.

[0182] Generation of iPSC and CRISPR-Cas9 gene editing: To generate isogenic lines, we introduced the G allele of SNP rs11154337 into control iPSCs, using pSpCas9(BB)-2A-GFP (PX458; Addgene; 48138) as a vector for genome editing. For site-specific CRISPR-Cas9 and guide RNA sequence construction, two reverse complementary guide oligonucleotides were annealed and connected to the linearized PX458 vector (Table 4). Single-stranded oligodeoxynucleotide (ssODN) templates with mutation / correction sites were designed as previously described (FARan et al., Genomeengineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308 (2013)). One day before transfection, control iPSCs were dispersed into single cells at 40% to 50% confluence. The next day, CRISPR-Cas9 and ssODN templates 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 in 6-well plates at a density of 2,000 cells per well. After one week of expansion, single-cell-derived colonies were observed and picked for genotyping and subsequent expansion (Table S4). DNA was extracted from the collected cells using QuickExtract solution (Epicenter). GXL DNA polymerase (Clontech) was used for PCR amplification and genotyping of the edited region.

[0183] Table 4 Oligonucleotides used for genome editing.

[0184]

[0185] Differentiation of iPSCs into endothelial cells: Generation of iPSC-ECs was accomplished using a chemical differentiation protocol with three major steps: mesoderm induction, endothelial cell 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) supplemented with B-27 (Life Technologies; A18956-01) without insulin. It is believed that the use of B27 without insulin can improve differentiation efficiency because insulin negatively affects mesoderm induction. Next, supplemented growth factors such as vascular endothelial growth factor (VEGF; 50 ng / mL; Gemini; 300196P) and fibroblast growth factor (FGF; 25 ng / mL; Gemini; 300113P) were used in EGM. TM -2 Endothelial Cell Growth Medium -2 BulletKit TM Endothelial lineage specification was performed in EGM. To increase the yield, the transforming growth factor β (TGFβ) inhibitor SB431542 (10 μM; Selleckchem; S1067) was also added because it promotes EC generation and inhibits smooth muscle cell differentiation from endothelial progenitor cells. Finally, iPSC-EC purification was performed using magnetic-activated cell sorting (MACS) targeting 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 by a column in a magnetic field, and then separated from unlabeled cells. Purified iPSC-ECs were maintained in EGM. TM -2 Endothelial Cell Growth Medium -2 BulletKit TM middle.

[0186] Characterization of iPSC-ECs by flow cytometry: CD144 + Cells before and after purification were analyzed for endothelial surface markers. Specifically, the mature endothelial progenitor cell marker CD34 (FITC anti-CD34 clone 581; BD Pharmingen TM ; 555821), mature endothelial markers 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 TM; 560872) expression (Table 3). Samples were analyzed by BD LSRFortessa at the Unified Flow Core of the University of Pittsburgh. TM The cells were run on a flow cytometer (BD Biosciences).

[0187] Characterization of iPSC-ECs by immunofluorescence staining: CD144 + After purification, iPSC-ECs 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 then blocked with 5% bovine serum albumin (BSA) for one hour at room temperature. Cells were stained with anti-VE-cadherin / CD144 antibodies (1:100; abcam; ab33168) or anti-CD31 (also known as platelet and endothelial cell adhesion molecule-1; PECAM-1; 1:100; abcam; ab24590) at 4°C overnight (Table 3). After rinsing with PBS, cells were incubated with appropriate secondary antibodies (1:1000) in 5% BSA for one hour at room temperature. Cells were rinsed with PBS and stained with ProLong containing DAPI. TM Gold Antifade Mountant (ThermoFisher; P36935) was used for mounting. Images were acquired on a Nikon A1 confocal microscope.

[0188] 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 extracts with reduced growth factors were plated in 96-well plates and allowed to solidify at 37°C for 30 minutes. iPSC-ECs were then plated (20,000 cells per well) in wells containing cell type-specific medium lacking growth factors and serum. Six hours later, capillary-like structures were imaged at 10× magnification using an EVOSTM XL core imaging system (ThermoFisher).

[0189] Transfected cells for RNA silencing: Human PAEC were transfected at approximately 70% to 80% confluence using negative control (siNC) or 20 nM target gene (siGene) silencing RNA (siRNA) (Table 5). 2000 (ThermoFisher; 11668019) was mixed with siRNA. The siRNA mixture was incubated with human PAEC in Opti-MEMTM reduced serum medium for 4 to 6 hours (ThermoFisher; 31985062). After incubation, the transfection medium was removed and replaced with full serum cell-specific growth medium. Experiments were performed 48 hours after transfection.

[0190] Table 5. Silencing RNA species.

[0191] siRNA gene targets Species <![CDATA[Silencer TM Assay ID]]> CH25H people 289454 NCOA7 people 242114 Negative control No. 1 people AM4611

[0192] 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 with HindIII and NheI linkers. The PCR products were directly cloned downstream of the green fluorescent protein (mGFP) open reading frame with a Myc tag in the pmGFP-ADAR1-p110 vector (Addgene; 117928). The cDNA sequences encoding full-length and short-length NCOA7 fused with mGFP were further subcloned into the pCDH-CMV-MCS-EF1α-Puro lentiviral expression vector (Systems Biosciences; CD510B-1). The cloned plasmids were verified by DNA sequencing at the Center for Genomics Research at the University of Pittsburgh.

[0193] HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. 2000 (ThermoFisher; 11668019) with lentiviral plasmids containing the target gene (or empty vector for control virus) and from ViraPower TM HEK293T cells were transfected with packaging plasmids from lentiviral packaging mix (ThermoFisher; K497500). Viral particles were harvested 48 hours after transfection, precipitated, and then filtered.

[0194] Cell transduction for lentiviral vector delivery: Human PAEC were transduced by direct application of polybrene (8 μg / mL) medium containing viral particles with either an empty control vector expressing GFP or with the target gene. Transduction efficiency was assessed by GFP expression as well as direct measurement of transcript and protein expression. Experiments were performed 72 hours after transduction.

[0195] Protein extraction and immunoblotting: Cells were washed twice with PBS and then collected in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the PierceTM BCA protein assay kit (ThermoFisher; 23225). 4-15% TGX TM Precast protein gel (Bio-Rad Laboratories; 4561086) separates protein lysates and then transfers them to PVDF membranes. The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for one hour at room temperature. Primary antibodies were then added and incubated overnight at 4°C (Table 3). The next day, the blots were washed three times with TBST for 10 minutes each. The blots were then incubated with appropriate secondary antibodies conjugated to HRP for one hour at room temperature. After another round of TBST washing, the blots were visualized using Pierce ECL reagents and images were captured using BioRad ChemiDoc XRS+.

[0196] Chromatin immunoprecipitation and quantitative polymerase chain reaction: MAGnify was used according to the manufacturer's protocol. TM Chromatin immunoprecipitation system (Invitrogen; 49-2024). Briefly, each ChIP reaction used 1×10 6 Personal PAEC or iPSC-derived EC. The cells were conjugated with rabbit IgG antibody (1 μg / μL) or rabbit NF-κB p65 antibody (5 μg / μL) at 4°C for one hour (Table 3). 6 Each cell was trypsinized, pelleted, and resuspended in 500 μL. Each reaction was crosslinked with 1% methanol-free formaldehyde for 10 minutes at room temperature. The crosslinking reaction was quenched with 1.25 M glycine for five minutes at room temperature. From this point on, the reaction was maintained at 4°C for all steps. The samples were pelleted and washed three times with cold PBS. Each reaction was then resuspended in 50 μL of lysis buffer containing protease inhibitors and chromatin shearing was performed. Cells were sheared with a UCD-200 using a 20-second ON and 40-second OFF protocol for six cycles. The samples were pelleted and the supernatant containing the chromatin product was collected and confirmed to have appropriate fragmentation by DNA gel. Incubate with chromatin for two hours at 4°C while rotating up and down. Then, wash the sample with a series of immunoprecipitation buffers and then reverse the cross-links with proteinase K. Then, purify the DNA and perform quantitative PCR. The primers used for ChIP-qPCR are listed in Table 6.

[0197] Table 6. Custom primers for ChIP qPCR.

[0198]

[0199]

[0200] Chromatin conformation capture (3C) assay: 3C assay was performed. Briefly, 1×10 7 Individual PAECs were crosslinked with 1% formaldehyde for 10 minutes at room temperature. Nuclei were isolated and genomic DNA was digested with 400U BspHI at 37°C, 950rpm overnight. Restriction enzyme digestion generated a 1,666bp genomic DNA fragment containing SNP rs11154337 and a 9,269bp DNA fragment containing the NCOA7 promoter based on the prediction of the putative BspHI cleavage sites along the genome. The digested DNA was diluted and ligated with or without 4000U T4 DNA ligase in a 16°C water bath for four hours, followed by reversal of the crosslinked DNA with 100μg proteinase K at 65°C overnight. Then, the DNA was isolated and purified. PCR was performed with selected primers to target the potential connection fusion sequence of the DNA fragment containing SNP rs11154337 and the fragment containing the NCOA7 promoter, and close to the BspHI site: (1) 50bp 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) 57bp from the BspHI site at the 5' end of the SNP rs11154337 fragment, 5'-GAA ATG CCA GGG ATTCCT TA-3' (reverse primer, (SEQ ID NO:29)). The amplified product produced a 107bp fragment, confirming the presence of the fusion sequence. The PCR products were separated by gel electrophoresis and analyzed by DNA sequencing.

[0201] Transcriptome analysis of human PAEC and data availability: Microarray data were acquired using the Affymetrix Clariom D Human Array at the Center for Genomics Research at the University of Pittsburgh. Microarray chips were performed in triplicate on four groups for a total of 12 samples. PAECs received either knockdown controls or knockdown controls for the gene NCOA7. In addition, each group was then placed under control conditions or further challenged with the proinflammatory cytokine IL-1β for 24 hours. The raw data were processed using the Bioconductor package in R language to generate a list of differentially expressed genes, which were selected using a Benjamini-Hochberg corrected P-value of less than 0.05 to minimize the false discovery rate (FDR). The following additional packages: oligo, limma, affycore tools, gplots, pd.clariom.d.human were used to analyze the microarray data. In general, the process of this program is used 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 graphs to show consistency between samples, adding annotations, setting up a comparison matrix to run statistical analysis between groups, generating a list of differentially expressed genes, filtering said list, building a heatmap to depict overall trends, and overlaying the generated gene list with our PH network. Gene set enrichment analysis (GSEA) was then performed on the identified differentially expressed genes using GeneOntology, REACTOME, KEGG, and BioCarta.

[0202] Proximity ligation assay: Use Proximity ligation assay (Millipore Sigma; DUO92102) was used to assess the direct interaction of NCOA7 with the V-ATPase subunit ATP6V1B2. TM Lab-Tek TM IIChamber Slide TM The cells were plated in a 4% paraformaldehyde system (20,000 per 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 with mouse anti-NCOA7 antibody (SantaCruz Biotechnology; sc-393427), rabbit anti-ATP6V1B2 antibody (abcam; ab73404), both antibodies, or no antibody overnight at 4°C (Table 3). The cells were then incubated with PLUS and MINUS probes for one hour, ligated for 30 minutes, and expanded for 100 minutes at 37°C. The cells were then stained with ProLong with DAPI. TMSlides were mounted with Gold Antifade Mountant (ThermoFisher; P36935). Images were acquired on a Nikon A1 confocal microscope at the University of Pittsburgh Biological Imaging Center.

[0203] Transmission electron microscopy: Human PAEC grown on tissue culture plasticware were fixed 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) at 4°C overnight. The monolayers were washed three times in PBS and then post-fixed in 1% osmium tetroxide, 1% Fe6CN3 aqueous solution for one hour. The cells were washed three times in PBS and then dehydrated through a 30-100% ethanol series with multiple changes of 4% ethanol. 812 embedding resin (Polysciences). 812 The capsule was inverted on top of the cells to embed the culture. The blocks were cured overnight at 37°C and then at 65°C for two days. The monolayer of cells was peeled off the coverslip and re-embedded for cross-sectional area analysis. Ultrathin cross-sections (60nm) of the cells were obtained on a Riechert / LeicaUltraCut E ultramicrotome, post-stained in 4% uranyl acetate for 10 minutes, and then post-stained in 1% lead citrate for 7 minutes. The sections were observed on a JEOL JEM-1400Flash transmission electron microscope at 80kV. Images were captured using a bottom-mounted AMT digital camera. The acquired micrographs were analyzed manually in a blind manner. Lysosomal area was quantified using Fiji.

[0204] Evaluation of lysosomal hydrolase activity: Lysosomal activity and function were evaluated using enzyme activity measurements. Human PAEC were seeded on glass coverslips and stained for all lysosomal measurements. For the LysoLive assay (Marker Gene Technologies, Inc.; M27745), the β-glucosidase-specific substrate GlucGreen was incubated at 5 μM in culture medium at 37°C for 30 minutes. The cells were washed three times with ice-cold PBS and then fixed with 4% PFA for 15 minutes at room temperature. ProLong with DAPI was used to stain. TM The slides were mounted with Gold Antifade Mountant (ThermoFisher; P36935).

[0205] For the SiR-lysosome assay (Cytoskeleton, Inc.; CYSC012), cell-permeable peptides conjugated to silicon rhodamine (SiR) dye were incubated in human PAEC as a measure of active cathepsin D. SiR-lysosomes were incubated with cells at 1 μM and with the calcium channel blocker verapamil at 1 μM for 30 minutes at 37°C to increase signal intensity. Cells were rinsed three times with ice-cold PBS, fixed with 4% PFA, and mounted as described above.

[0206] Assessment of lysosomal acidification: using LysoSensor TM Lysosomal acidification was measured using yellow / blue DND-160 (PDMPO) dye (ThermoFisher; L7545). LysoSensor TM The yellow / blue DND-160 (PDMPO) dye exhibits dual excitation (i.e., 329 and 384 nm) and dual emission (i.e., 440 and 540 nm) spectral peaks that are pH-dependent (pK a 4.2). In acidic organelles, the dye fluoresces primarily in yellow. In alkaline organelles, the dye fluoresces primarily in blue. The unique spectral properties of this dye allow for ratiometric quantification.

[0207] Live human PAEC were incubated with 1 μM dye in 0.1% FBS cell-specific medium for 1 min at 37 °C. The cells were rinsed with PBS, trypsinized, pelleted at 300 g for 5 min in polystyrene tubes, and rinsed twice more with PBS. TM Cells were analyzed on a flow cytometer (BD Biosciences). The median fluorescence intensity (MFI) ratio was calculated using the ratio of the yellow MFI fluorescence value to the blue MFI fluorescence value.

[0208] Assessment of lysosomal lipid content: Neutral lipids were detected using the fluorescent dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-borono-3a,4a-diaza-s-indenene ( ThermoFisher; D3922). Acidic organelles (i.e., lysosomes) were stained with LysoTracker TM Red DND-99 (ThermoFisher; L7528) staining. Live human PAEC were incubated with 1 μM and 50 nM LysoTracker TMThe cells were incubated in cell type specific medium containing Red DND-99 at 37°C for 30 minutes. Then, the cells were washed three times with PBS and fixed with 4% PFA for 30 minutes at room temperature. The cells were rinsed three more times with PBS and then fixed with ProLong with DAPI. TM The slides were mounted with Gold Antifade Mountant (ThermoFisher; P36935). Images were acquired on a Nikon A1 confocal microscope at the University of Pittsburgh Biological Imaging Center.

[0209] pass (i.e. neutral lipids) and LysoTracker TM Lysosomal lipid content was measured by the degree of colocalization between Red DND-99 (i.e., acidic organelles). Colocalization was measured using EzColocalization in Fiji and quantified as Pearson's correlation coefficient.

[0210] Targeted LC-MS for cholesterol intermediates and oxysterols: Human PAECs were processed and collected, 1 × 10 6 cells for cholesterol intermediate and oxysterol analysis. The cells were subjected to liquid-liquid extraction of sterols. Briefly, 1 mL of dichloromethane, methanol, and water were added to each sample. The samples were then vortexed and centrifuged to produce two liquid phases. The lower phase was carefully transferred to a new glass tube and dried under nitrogen. The samples were then resuspended in hexane and the resulting lipid species were analyzed by liquid chromatography-mass spectrometry (LC-MS). The samples were run on a SCIEX QTRAP 6500+ equipped with a Shimadzu LC-30AD HPLC system and a 150×2.1-mm, 5μm Supelco Ascentis silica gel column. LC-MS data were analyzed using MultiQuant (SCIEX).

[0211] Statistical Analysis of the UPMC and STRIDE Cohorts: For the UPMC cohort, the effect of the SNP rs11154337G minor allele on six-minute walk distance was calculated using a dominant inheritance model. In addition, the effect of the minor allele on time to death or last follow-up was tested using a Cox proportional hazards model. In both models, genetic effects were adjusted for sex, age, comorbidities, and vasodilator therapy. These analyses were performed using Stata 17.0 pymol.

[0212] For the STRIDE cohort, analyses were performed starting with raw data in plink format. The strand information was converted to InfiniumOmniExpress-24v1-3_A1-b37.strand using update_build.sh. Next, phase and impute2 were performed using shapeit and 1000G_Phase3 b37 for imputation. Chromosomal bins were merged and redundant SNPs were removed using gtool. The first survival analysis was performed using the R package gwasurvivr, with a coxph test for each SNP relative to the time of death (FinalEvent), including the covariates: sex, age, PAH type, WHO classification, study inclusion (Encysive or Prospective), AnyDrugsBefore, UsePDE, UsePros, UseWarf, UseOxy, and a maf filter of 0.005. Further analyses were performed testing SNP rs11154337 and time to death (FinalEvent) in patients of European ancestry (EthConEUR) based on self-reported ethnicity and discriminant principal component analysis.

[0213] Neutral lipid staining: Use the fluorescent dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-boron-3a,4a-diaza-s-indenene Neutral lipid staining in cells. Live cells were grown on glass coverslips in plastic dishes. Cells were rinsed three times with PBS to remove residual culture medium. 2 μM The staining solution was applied to the cells for 15 min at 37 °C. The cells were rinsed three times with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature. ProLong with DAPI was used. TM Coverslips were mounted with Gold Antifade Mountant (ThermoFisher; P36935). Images were acquired on a Nikon A1 confocal microscope at the University of Pittsburgh Biological Imaging Center.

[0214] Measurement of cholesterol uptake: To assess cholesterol uptake, a cholesterol uptake assay kit was used according to the manufacturer's instructions (abcam; ab236212). Briefly, treated human PAECs were incubated for 24 hours in cell type-specific medium supplemented with fluorescent NBD-cholesterol (at 20 μg / mL) in 0.1% serum. Cells were rinsed with PBS, trypsinized, pelleted at 300 g for 5 minutes in a polystyrene tube, and rinsed twice more with PBS. Immediately, BD LSRFortesssa at the University of Pittsburgh's Unified Flow Cytometry Center was used to analyze the cells.TM Cells were analyzed on a flow cytometer (BD Biosciences). Flow cytometry analysis was chosen over confocal microscopy due to the high rate of photobleaching observed with NBD cholesterol.

[0215] Cholesterol Level Assessment: To assess cholesterol levels, use Cholesterol / Cholesterol Ester-Glo TM Assay kit (Promega; J3190). The assay measures cholesterol using cholesterol dehydrogenase, which links the presence of cholesterol to NADH production and thus to luciferin activation. Human PAEC were seeded in 96-well plates at a density of 20,000 cells per well and replicated six times. The assay was performed according to the manufacturer's instructions.

[0216] Measuring apoptosis by caspase-3 / 7 activity: using Apoptosis was assessed using the 3 / 7 assay system (Promega; G8090). The assay works by providing a luminescent caspase-3 / 7 substrate optimized for caspase activity. Cleavage of the substrate generates a luminescence-based signal by luciferase. An equal volume of the substrate was added to wells containing human PAEC (5,000 per well) and incubated at room temperature for 30 minutes. Luminescence was measured spectrophotometrically. Luminescence signals were normalized to protein content per well using Pierce TM The protein was evaluated using the BCA protein assay kit (ThermoFisher; 23227).

[0217] Proliferation was measured by BrdU incorporation: Proliferation was assessed using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology; 6813). The assay works by measuring BrdU incorporation into proliferating cells using an anti-5-bromo-2'-deoxyuridine BrdU antibody. BrdU was added to complete growth medium containing 5% serum for two hours. Human PAECs (5,000 per well) were fixed and denatured before applying mouse anti-BrdU antibodies. Next, anti-mouse HRP-linked antibodies were added. Then, a chromogenic substrate was added to detect with the antibody complex of HRP-linked BrdU. The absorbance was measured at 450 nm using spectrophotometry.

[0218] Leukocyte and monocyte adhesion assay: To assess immune activation of the endothelium by measuring the adhesion of immune cells to the endothelial monolayer. Human PAECs were cultured until a complete monolayer was formed. CellTrace TMBlue or CFSE (ThermoFisher; Blue, C34568; CFSE, C34554) were used to stain immune cells. 5 Each stained immune cell was added to each well of a six-well plate and allowed to incubate for 24 hours. The wells were then rinsed twice with PBS and subsequently fixed with 4% PFA for 15 minutes at room temperature. After fixation, the cells were rinsed again with PBS. Fluorescence images of each well were acquired at 4× magnification. The number of immune cells in each image was quantified using Fiji. For leukocyte adhesion assays, HuT78 skin T lymphocytes (ATCC) were used. For monocyte adhesion assays, THP-1 peripheral blood mononuclear cells (ATCC) were used.

[0219] Application of oxysterols and bile acids: Oxysterols and bile acids were applied to human PAEC in 0.1% FBS cell-specific medium. 25-Hydroxycholesterol and 7HOCA were dissolved in 100% ethanol and applied to cells at a concentration of 25 μM or 50 μM, respectively, for 24 hours.

[0220] Animal studies: All animal studies were approved by the Department of Laboratory Animal Resources at the University of Pittsburgh. The Ncoa7 knockout mouse line (C57BL / 6Ncoa7tm1.1 (KOMP) Vlcg) was obtained from the Knockout Mouse Project (KOMP; komp.org) and re-derived using sperm at the Genome Editing, Transgenic, & Virus Core at the Magee Women's Research Institute. The obtained mice were bred in-house to generate homozygous Ncoa7 knockout mice. To obtain a model of lung inflammation that causes severe PH, Ncoa7 knockout mice were crossed with C56BL / 6Il6 transgenic (Tg + ) mice. +Mice contain the Clara cell 10-kD promoter (CC10), which drives constitutive expression of IL-6 in the lung (MK Steiner et al., Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104, 236-244, 228p following 244 (2009)). C57BL / 6 mice were used to deliver PBS or 7HOCA (10 mg / kg) continuously through the orotracheal tube every 5 days for 4 weeks under chronic hypoxia (10% O2). Mice were raised under normal oxygen tension until 15 weeks of age, and then echocardiography, invasive hemodynamic measurements and tissue collection were performed. The PAH rat monocrotaline model was utilized by a single injection of monocrotaline (80 mg / kg) at 8 to 9 weeks of age. After the monocrotaline loading dose, rats were injected intraperitoneally with DMSO or 958 ami (7.5 mg / kg) for 10 days and then discontinued.

[0221] Hemodynamic measurements: Echocardiography was performed on 15-week-old mice using a 15-45 MHz transthoracic transducer and a VisualSonics Vevo770 system (Fujifilm). During animal positioning and hair removal, 2% isoflurane in 100% O2 was administered for anesthesia, which was then reduced to 0.8% isoflurane during image acquisition. Data were analyzed by technicians in a blinded manner.

[0222] For right heart catheterization, mice were given ketamine / xylazine (9:1; Henry Schein) or mice received isoflurane (Henry Schein). The isoflurane vaporizer was maintained at 1.5 to 2% with an oxygen 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 through the right atrium and into the right ventricle. Steady-state right ventricular systolic pressure waveforms were measured for two minutes. Waveform analysis was performed in a blinded manner.

[0223] NCOA7 短 Structural modeling of the catalytic domain of NCOA7 短 The sequence of (subtype 5; Q8NI08-5) was downloaded from UniProt. The catalytic domain (P55-D219) was modeled using SWISS-MODEL based on the crystal structure of the TLDc domain of zebrafish antioxidant protein 2 (OXR2) (PDB ID4ACJ). The sequence identity between NCOA7 and OXR2 catalytic domains was calculated to be 61.8% using Clustal Omega, indicating that the two domains have the same structure.

[0224] Gaussian Network Model (GNM) Analysis: In GNM analysis, protein structures are represented as elastic networks with residues as nodes whose positions are identified by those of the α-carbon. Therefore, a total of 165 residues were used to develop the GNM of NCOA7. The total potential energy is represented as the sum of the harmonic potentials between pairs of nodes within the interaction range, which is defined as C α -C α Distance less than The resulting topology of the network is recorded in the NXN Kirchhoff matrix. All calculations are performed using the ProDy API.

[0225] Drugability simulations and analysis: Drugability simulations of NCOA7 were performed in the presence of probe molecules using the all-atom MD simulation package NAMD, employing the CHARMM36 force field for proteins, the TIP3P water model, and the CGenFF force field for probe molecules. The probe molecules were benzene, isobutane, imidazole, acetamide, isopropanol, isopropylamine, and acetate, and were derived from a statistical evaluation of the most common chemical / functional groups in FDA-approved drugs. Trajectories were analyzed using the DruGUI module of ProDy, and six independent runs of 40 ns were performed. All MD snapshots were acquired using C α - Atomic and spatial superposition of cubic grid representation onto the reference PDB structure, with the grid edge size set to The distance between the non-hydrogen atoms of the probe molecule and the protein atoms is Within , they are considered to interact with the protein. For each probe type, individual occupancies of the grid were calculated using their centroids. We evaluated the occupancy of each voxel for each probe and quantified the binding energy using the inverse Boltzmann relation. The resulting binding free energy map identifies interaction points where one or more probe types have low (favorable) energy, and high occupied voxels are called druggable hotspots.

[0226] Pharmacophore modeling: Using Pharmmaker, we identified residues involved in high-affinity interactions with each molecular probe type. Residue-probe interactions were then ranked based on their frequency of occurrence at druggable hotspots in multiple runs. Snapshots that simultaneously exhibited residue-probe pairing probabilities (e.g., N62-benzene, E66-isopropylamine, P80-benzene, and W81-benzene) were selected as templates to construct pharmacophore models. The pharmacophore model contained hydrogen bond donors and hydrophobic features at the isopropylamine site and hydrophobic and aromatic rings at the two benzene sites. Pharmit was then used to screen the pharmacophore model against the ZINC and MolPort libraries. The MolPort library contains 67,033,884 conformations corresponding to 4,848,718 compounds, and the ZINC library contains 122,276,899 conformations corresponding to 13,127,550 compounds. Among the top-scoring compounds, MolPort-004-267-958 was selected for further refinement after preliminary experimental validation.

[0227] 958 and 958 ami Molecular dynamics simulations: All atomic MD systems were set up using the GHARMM-GUI SolutionBuilder. The simulations were performed using the MD simulation package NAMD, using the CHARMM36m force field for proteins, the TIP3P water model, and the CGenFF force field for compounds. ami Three independent runs were performed, each of 0.2 ms (total 0.6 ms). In each case, the system contained NCOA7 protein and compound, and relaxation was performed using the equilibrium step in CHARMM-GUI in the presence of significant water and 0.15 M NaCl. We performed NPT dynamics for 0.2 ms with a time step of 2 fs and constant pressure (1 bar) and temperature (300 K). Contact time and hydrogen bond formation between target and compound 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.

[0228] Statistics: All in vitro data represent at least three independent experiments. The number of animals used for a given experimental model was calculated to measure at least 20% difference between the mean values ​​of the control group and the experimental group, with an efficacy of 80% and a standard deviation of 10%. The number of patient samples used for molecular analysis was mainly determined by clinical availability. The Shapiro-Wilks test was used to determine the normality of the data distribution. For normally distributed data, paired data were analyzed with a two-tailed Student t-test, and grouped data were compared using one-way or two-way analysis of variance combined with post hoc Tukey analysis to correct for multiple comparisons. Significance was determined by a P value of less than 0.05. All data are expressed as mean ± standard deviation.

[0229] result

[0230] Convergent inflammatory regulation of NCOA7 in cellular, animal, and human examples of PAH: Unbiased transcriptome analysis of primary human pulmonary artery endothelial cells (PAECs) exposed to IL-1β, a proinflammatory cytokine elevated in the plasma of PAH patients and known to be a trigger for disease pathogenesis. Lysosomal regulatory genes were upregulated overall, with distinct subsets containing V-ATPase subunits, which are binding partners of NCOA7 that promote lysosomal acidification ( Figure 4 (A) ). Accordingly, IL-1β up-regulated NCOA7, the canonical full-length isoform (NCOA7 全长 ) and to a greater extent the alternative-start, short-length version of NCOA7 (NCOA7 短 )( Figure 4 (B, C), Figure 5A (A)).

[0231] Other triggers of EC dysfunction in PAH similarly upregulate NCOA7. Specifically, exposure to the proinflammatory cytokine IL6 and its soluble receptor (IL6Rα), which has been linked to PAH, induces expression of both the short and full-length isoforms ( Figure 5A (B, C)). Hypoxia, a known driver of PH, also increased both isoforms ( Figure 5A (D, E)). Taken together, these data suggest that NCOA7, particularly its unique alternative initiation isoform, plays a potential role in multiple PH triggers.

[0232] Using a severe inflammatory rodent model of PAH, transgenic mice under constitutive IL-6 overexpression and chronic hypoxia showed that CD31 isolated from lung tissue + Ncoa7 expression is increased in EC ( Figure 5AThis was also observed in the pulmonary vasculature of Il6 transgenic mice without hypoxia (a mild form of experimental PAH) ( Figure 4 (D, E)). Similarly, in the chronic hypoxia mouse model and the monocrotaline-exposed PAH rat model, NCOA7 was significantly upregulated in the pulmonary vessels, and its expression in the endothelium was enhanced ( Figure 5A (HK)).

[0233] Furthermore, human lung tissue from group I PAH patients showed elevated NCOA7 expression within pulmonary vasculature compared with healthy controls ( Figure 4 (F, G)). Single-cell RNA sequencing of the lungs of patients with idiopathic group I PAH revealed an increase in the number of ECs expressing NCOA7 compared with healthy controls (22.58% vs. 29.66%) ( Figure 4 (H)). In addition, in NCOA7-positive ECs, NCOA7 expression was upregulated in PAH patients ( Figure 4 (I)). Thus, in proinflammatory models of PH using primary PAEC, rodent models, and human patients, we found that NCOA7 is upregulated within pulmonary vasculature, most importantly within endothelial cells. However, because NCOA7 deficiency and the resulting loss of lysosomal acidification increases inflammation and exacerbates disease in other contexts, NCOA7 is thought to act as a homeostatic brake under proinflammatory stress to reduce disease pathogenesis by attenuating EC immune activation.

[0234] To investigate the upstream inflammatory mechanisms regulating NCOA7, we predicted binding sites for the RelA / p65 (RELA) subunit of the established inflammatory transcription factor complex, NF-κB, in the canonical (i.e., full-length) and atypical (i.e., short-length) NCOA7 promoter regions. Accordingly, knockdown of RELA in PAECs abolished IL-1β-mediated upregulation of both isoforms ( Figure 4 (J, K)). Chromatin immunoprecipitation combined with quantitative PCR (ChIP-qPCR) showed that RelA / p65 was significantly enriched in DNA sequences of typical and non-typical promoters ( Figure 4 (L) ), indicating direct promoter-transcription factor binding.

[0235] NCOA7 deficiency promotes lysosomal dysfunction and lipid accumulation: To investigate the putative link between NCOA7 and oxysterol production in the presence of proinflammatory conditions, we characterized NCOA7-mediated control of lysosomal acidification given the known function of lysosomes in sterol trafficking. In human PAECs exposed to IL-1β, NCOA7 knockdown reversed interleukin-specific alterations in the gene network controlling lysosomal function ( Fig. 6A (A) Notably, many of these genes (e.g., ATP6V0A1, ATP6V1B2, ATP6V1C1, ATP6V1D, ATP6V1E1, ATP6V1G1, and ATP6V1H) encode subunits of the V-ATPase machinery that are essential for lysosomal acidification and, therefore, for the function of the pH-sensitive enzyme.

[0236] Proteomics-based studies determined that NCOA7 interacts with ATP6V1B1, a kidney-specific paralog of ATP6V1B2. Accordingly, in PAECs, NCOA7 knockdown abolished IL-1β-mediated upregulation of ATP6V1B2, and forced overexpression of either the short or full-length isoforms upregulated ATP6V1B2 ( Fig. 6A (B, C)). In addition, ATP6V1B2 is upregulated in the lung endothelium of rodent and human PH models ( Figure 7 A and 7B (AH)). To assess direct interactions between these proteins, proximity ligation assays revealed perinuclear staining indicative of ATP6V1B2-NCOA7 interaction and consistent with perinuclear distribution in lysosomes. 短 or NCOA7 全长 Forced overexpression of upregulated the amount of ATP6V1B2-NCOA7 interaction in lysosomes ( Fig. 6A (D, E)). These data suggest that both short and full-length NCOA7 function as regulatory components of the V-ATPase complex with a putative downstream lysosomal function.

[0237] To assess the activity of NCOA7 in regulating lysosomal acidification, we utilized two quantitative measures of lysosomal enzyme activity. First, IL-1β increased cleavage of the fluorescent LysoLive tracer, which was subsequently blocked by NCOA7 depletion ( Figure 6B (F, G)). Second, lysosome-dependent cathepsin D activity was assessed using SiR-lysosomes. Consistent with the IL-1β-mediated increase in ATP6V1B2, IL-1β significantly upregulated cathepsin D activity, as confirmed by SiR-lysosome fluorescence, and NCOA7 knockdown abolished this activity ( Figure 6B (H, I)).

[0238] To directly assess lysosomal acidification, the acidotropic probe LysoSensor Green DND-189 was utilized, which accumulates in acidic compartments and enhances fluorescence under acidic conditions. Consistent with the IL-1β-mediated increase in lysosomal enzyme activity, IL-1β increased the LysoSensor fluorescence signal, while NCOA7 knockdown reversed this ( Figure 6B(J)). In addition, when the acidotropic probe LysoSensor Yellow / Blue DND-160 was used, IL-1β induced a shift to yellow fluorescence in PAEC, indicating enhanced acidification of the lysosomal lumen ( Figure 6B (K)). Addition of NCOA7 deficiency reverses the IL-1β-mediated yellow fluorescence shift. These observations mimic the findings in LSD, which are notable for the accumulation of undigested cellular components in the lysosomal compartment.

[0239] Failure of this V-ATPase complex formation or lysosomal acidification is a known driver of abnormal lysosomal morphology. Therefore, morphological analysis of lysosomes in NCOA7-deficient human PAECs using transmission electron microscopy revealed marked hypertrophy of lysosomes, as measured by lysosomal area quantification ( Figure 6B (L, M)), indicating that the lysosomal compartment is unable to break down and process cellular components. In addition, enlarged lysosomes in NCOA7-deficient cells carry lamellar inclusions ( Figure 6B (L, M); yellow arrows), indicating lipid accumulation—again phenocopying LSDs that exhibit aberrant lysosomal lipid accumulation.

[0240] To determine whether the lamellar structures observed in the electron micrographs were lipids, a dye specific for neutral lipids (i.e. ) and a dye that specifically localizes to the acidic compartment (i.e., LysoTracker) were co-stained in PAEC. With the loss of NCOA7, lipid punctae increased throughout the cell, suggesting vesicular accumulation. Accordingly, high-intensity lipid punctae were identified within acidic vesicles, supporting the view that the loss of NCOA7 leads to lysosomal lipid accumulation ( Figure 6C (N, O)). Taken together, these findings identify NCOA7 as a binding partner of the V-ATPase complex to promote lysosomal acidification and sterol trafficking in PAECs.

[0241] NCOA7 deficiency reprograms sterol metabolism through aberrant lipid accumulation: alterations in lysosomal lipid trafficking affect sterol homeostasis. Correspondingly, transcriptome analysis of NCOA7-deficient human PAECs revealed a marked enrichment and downregulation of biosynthetic processes associated with sterol metabolism ( Fig. 8A (A, B); red arrows). In sterol-saturated cells, the extracellular cholesterol uptake pathway is inhibited, specifically by reducing the density of low-density lipoprotein receptors (LDLR) on the cell membrane. NCOA7 deficiency in PAECs reduces LDLR expression ( Fig. 8A (C) ), accompanied by a functional reduction in the uptake of fluorescently labeled cholesterol ( Fig. 8A(D, E)). Total cholesterol levels were also upregulated in NCOA7-deficient PAECs, whereas forced overexpression of NCOA7 reduced total cholesterol levels ( Fig. 8A (F, G)). No obvious difference in post-squalene intermediates was detected between NCOA7-deficient PAECs and NCOA7-sufficient PAECs ( Fig. 9A and 9B (AN)), indicating that NCOA7-dependent regulation of sterol intermediate flux is independent of de novo cholesterol synthesis. Thus, the downregulation of sterol metabolism caused by NCOA7 deficiency is primarily caused by altered lysosomal sterol processing rather than de novo synthesis.

[0242] To prevent cholesterol accumulation, cells can either export cholesterol directly through transporters or increase the solubility of cholesterol through a series of oxidation steps. Thus, NCOA7 deficiency significantly upregulates cholesterol 25-hydroxylase (CH25H), an oxysterol-generating enzyme that increases cholesterol solubility ( Fig. 8A (H)). CH25H is upregulated in the pulmonary vessels of proinflammatory rodent models of PAH and group I PAH patients and localizes to the endothelium ( Figure 8B (IN) and Fig. 9B (O, P)), showing the in vivo relevance of these processes. Collectively, these data establish that NCOA7 plays a central role in maintaining EC sterol homeostasis by generating oxysterol species in cultured PAEC and in diseased endothelium in vivo.

[0243] NCOA7 deficiency induces endothelial production of oxysterols and downstream bile acid derivatives: To confirm whether the observed upregulation of CH25H enhances oxysterol production, targeted lipidomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS). NCOA7 knockdown in human PAEC exposed to IL-1β significantly upregulated 25-hydroxycholesterol (25HC), 27-hydroxycholesterol (27HC), and autoxidatively generated 7α-hydroxycholesterol ( Figure 8B (OQ)). These oxysterols are known to be metabolized into downstream bile acid derivatives through mechanisms that are not yet fully understood. Thus, NCOA7 knockdown 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-pentanol ( Figure 8B (RT)). In addition, the upstream metabolites 3β,7α-dihydroxy-5-cholestenoate and 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) were also upregulated ( Figure 8B (V, W)). Therefore, it is consistent with the upregulation of oxysterol-producing enzyme CH25H ( Fig. 8A(H) ), NCOA7 deficiency induces the production of abundant oxidized cholesterol metabolites and downstream bile acids in ECs.

[0244] Oxysterols and bile acids as markers of morbidity and mortality in PAH: highlighting the clinical importance of this mechanism in controlling disease severity, identifying plasma signatures including the same NCOA7-dependent sterols and bile acids that were associated with mortality in PAH (adjusted P < 1.1 × 10 -6 In a companion study, an unbiased, metabolome-wide association study of the multicenter PAH Biobank cohort (N=2,796) identified 13 different plasma oxysterols and bile acids that most effectively predicted four-year mortality in PAH. Notably, of these top 13 oxysterols and bile acids, four were upregulated in NCOA7-deficient ECs ( Figure 8B (Q, R, S, W)) and upregulated in the serum of Ncoa7-deficient mice ( Fig.13 These findings highlight the clinical importance of NCOA7-dependent oxysterols and bile acids in controlling PAH severity, establishing a framework linking lysosome-dependent inflammation to population-level and metabolome-wide signaling in PAH.

[0245] NCOA7 deficiency promotes endothelial dysfunction via oxysterol production: Given the immunoregulatory functions of oxysterols in diseased endothelium, we sought to determine whether NCOA7 deficiency promotes EC dysfunction in an oxysterol-dependent manner. NCOA7 deficiency upregulates vascular cell adhesion molecule 1 (VCAM1), a surrogate for endothelial immune activation ( Fig. 10A (A, B)). In contrast, forced overexpression of NCOA7 isoforms reversed VCAM1 expression ( Fig. 10A (C, E)). To determine whether NCOA7 deficiency is dependent on downstream oxidized forms of cholesterol to induce PAEC pathological phenotypes, concomitant knockdown experiments were performed on the oxysterol-generating enzyme CH25H, which is upregulated by NCOA7 deficiency. Notably, inhibition of CH25H induction in the presence of NCOA7 deficiency prevented VCAM1 expression ( Fig. 10A (E, F)).

[0246] Consistent with the changes in VCAM1 observed in various states of NCOA7 and CH25H expression, we observed increased attachment of leukocytes and monocytes to NCOA7-deficient EC monolayers ( Fig. 10B (G, J) ), which can be reversed by NCOA7 overexpression ( Fig. 10B (H, K)), and in the case of NCOA7 deficiency, inhibition of CH25H significantly attenuated immune cell attachment ( Fig. 10B (I, L)).

[0247] Furthermore, and consistent with the concept of immune activation, anti-apoptotic, and hyperproliferative endothelium in pulmonary vascular disease, NCOA7 deficiency in PAEC abolished IL-1β-mediated apoptosis while enhancing proliferation ( Fig.11A (A, D)). NCOA7 promotes PAEC apoptosis under pro-inflammatory conditions ( Fig.11A (B) ), while attenuating proliferation, which was more pronounced under IL-1β ( Fig.11A (E)). However, inhibition of CH25H upregulation reversed the attenuation of apoptosis and enhanced the proliferation capacity of cells ( Fig.11A (C, F)). In conclusion, the presence of NCOA7 prevents immune activation of the endothelium, thereby inducing apoptosis and inhibiting proliferation.

[0248] To determine whether bile acids are sufficient to immunologically activate the endothelium, 7HOCA was applied directly to cultured PAECs. 7HOCA displayed its pro-inflammatory properties, significantly upregulating VCAM1 ( Fig. 10C (M, N)), thereby promoting the adhesion of both leukocytes and monocytes to the PAEC monolayer ( Fig. 10C (O, P)). Notably, direct application of 25HC and downstream derivatives such as triols and tetraols similarly upregulated VCAM1 and enhanced immune cell adhesion to PAEC monolayers ( Fig. 11B Together, these findings suggest that oxysterol-generating enzymes and their downstream oxysterol species are necessary and sufficient to mediate immune activation in NCOA7-deficient lung endothelial cells.

[0249] NCOA7 deficiency and orotracheal delivery of 7HOCA exacerbate PAH in vivo: To determine whether the presence of NCOA7 prevents EC immune activation in PAH severity, we used an Il6 transgenic (Tg + ) mice induced severe lung inflammation as a model of angiogenic PAH (MKSteiner et al., Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104, 236-244, 228p following 244 (2009)). Ncoa7 knockout mice were combined with Il6Tg + mice to determine whether NCOA7 deficiency exacerbates PH in vivo ( Fig.14 (A)). Echocardiographic evaluation excluded any significant changes in left ventricular function as shown by left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and left ventricular posterior wall distance during diastole and systole (LVPW; d and LVPW; s) ( Fig.13 A(AE)).

[0250] Ncoa7-deficient mice exhibited elevated CH25H expression in pulmonary arterioles, accompanied by elevated plasma levels of 7HOCA and tetraols ( Fig.14 (B, C, G) and Fig.13 (FI)). These findings are consistent with the oxysterol and bile acid plasma profiles associated with PAH severity in humans and with our studies in cultured PAECs. Elevated 7HOCA in Ncoa7-deficient mice leads to endothelial immune activation, as evidenced by enhanced expression of VCAM1 and CD11b + The infiltration of mononuclear cells ( Fig.14 (BE)). In addition, Ncoa7-deficient mice showed increased muscularization of pulmonary arterioles ( Fig.14 (F) ), with worsening hemodynamic manifestations of PAH and increased right ventricular systolic pressure (RVSP) and Fulton index (a measure of right ventricular remodeling) ( Fig. 12A (B, I)).

[0251] Finally, to directly assess the pathogenicity of 7HOCA, we utilized a mouse model of chronic hypoxia by continuous orotracheal delivery of saline or 7HOCA ( Fig.14 (J) ), and echocardiography confirmed that left ventricular function was unchanged ( Fig.13 (JN) As expected, orotracheal delivery of 7HOCA upregulated endothelial immune activation, as evidenced by enhanced expression of VCAM1 and CD11b + Monocyte infiltration into the vascular bed is shown in ( Fig.14 (KM)). Consistent with Ncoa7 knockout, 7HOCA exacerbated PAH, as reflected by increased pulmonary arteriolar remodeling and increased RVSP ( Fig. 12B (N, P)). In summary, our data indicate that genetic deletion of Ncoa7 or direct delivery of the proinflammatory sterol 7HOCA is sufficient to promote PH in vivo.

[0252] Intronic SNP rs11154337 controls RelA / p65 binding to the NCOA7 atypical promoter and is associated with PAH disease severity and mortality: Thus, these data so far define NCOA7 as a homeostatic brake under proinflammatory conditions that maintains lysosomal activity and sterol trafficking to attenuate EC immune activation in PAH. Based on the role of single nucleotide polymorphisms (SNPs) in regulating gene promoter activity, we sought to determine whether pathogenic NCOA7 defects might result from inherited SNP-dependent control of NCOA7 expression and its downstream functions. First, annotated SNPs were investigated based on their proximity to both canonical and atypical promoters and high levels of epigenetic marks indicative of elevated transcriptional activity. In this process, we found a candidate SNP, rs11154337, which is located near the intronic region proximal to the atypical promoter of NCOA7 and carries a large number of histone modifications (WJ Kent et al., The human genome browser at UCSC. Genome Res 12, 996-1006 (2002)). Secondly, given the tandem regulation of the short and long subunits of NCOA7 in PAH ( Figure 4 ), attempting to discern any potential regulatory function of this SNP by positional backfolding to a typical promoter. To this end, human umbilical vein endothelial cells (GEO ID GSM3438650 and GSM3438651) were subjected to publicly available high-throughput chromatin conformation capture (3C) in the 3D genome interaction viewer and database (3DIV) (D. Yang et al., 3DIV: A 3D-genome Interaction Viewer and database. Nucleic Acids Res 46, D52-D57 (2018)). These maps showed that SNP rs11154337 had a long-range interaction of more than 120 kilobases with the typical promoter region of NCOA7 ( Fig.15A (A)). To establish this interaction, we performed 3C on human PAEC and found that the 3' end of the restriction enzyme-digested DNA fragment containing the NCOA7 transcription start site (N3) was ligated to the 5' end of the digested fragment containing SNP rs11154337 (S5) to generate a nucleotide fusion PCR product (N3S5) that was confirmed by sequencing ( Fig.15A (B, C)). In addition, ChIP targeting RelA / p65 revealed a significant enrichment in the region containing the SNP, indicating the presence of a p65 protein-SNP complex ( Fig.15A (D)).

[0253] Given the control of NCOA7 gene structure by SNP rs11154337, combined with the finding that NCOA7 controls an oxysterol signature associated with PAH severity and mortality, we sought to determine whether this SNP was also associated with disease severity and mortality using two independent PAH cohorts. Fig. 12A (AC)). First, a single-center PAH cohort of European ancestry 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 six-minute walk distance (P = 0.0130; β = 66.90, 95% CI [14.45-119.36]), Fig. 12A (A)). Importantly, after adjustment for age, sex, and use of vasodilators, patients carrying the homozygous G allele had significantly increased survival (P = 0.0250; hazard ratio = 0.44, 95% CI [0.21-0.90], Fig. 12A (B)). In a second multicenter PAH cohort of European descent from the Sitaxsentan To Relieve Impaired Exercise (STRIDE) trial from 45 centers in the United States and Canada (STRIDE, N = 63 (RLBenza et al., Endothelin-1Pathway Polymorphisms and Outcomes in Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 192, 1345-1354 (2015)), the presence of the G allele was confirmed to confer a survival benefit (P = 0.0002, hazard ratio = 0.49, 95% CI [0.34-0.71], Fig. 12B (C)). Thus, analysis of genomic, metabolomic, and clinical datasets from a PAH patient cohort revealed reciprocal activities of NCOA7 and SNP rs11154337 with glucuronidation of oxysterols and clinical outcomes of PAH.

[0254] SNP rs11154337 regulates NCOA7 and its downstream pathogenic functions: Using this concept—and guided by the negative association of the G allele of the NCOA7 intronic SNP rs11154337 with an oxysterol signature predictive of mortality and clinical measures in PAH—we sought to determine whether this SNP controls NCOA7 expression, lysosomal activity, and the production of oxysterol and bile acid metabolites, thereby regulating EC behavior. To investigate the cellular and biological activities of SNP rs11154337 embedded near the atypical NCOA7 promoter, a panel of genetically matched isogenic induced pluripotent stem cell (iPSC) lines (C / C versus C / G genotypes, respectively) were generated by CRISPR-Cas9 gene editing with allelic variants of SNP rs11154337. Fig. 12A (D) and Fig.15A (F)). iPSCs were then 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)). Purified iPSC-ECs showed significant enrichment of EC markers CD34, CD144, and CD309, and immunofluorescence staining of iPSC-ECs for CD144 and CD31 showed a pattern consistent with endothelial ( Fig. 15B (G, H)). In addition, iPSC-ECs showed angiogenic potential, as shown by blood vessel formation in growth factor-depleted Matrigel ( Fig. 15B (H)).

[0255] Compared with the C / C line, C / G iPSC-ECs showed higher expression of both short and long NCOA7 isoforms, confirming that the G allele increases NCOA7 transcription ( Fig. 12A (E, F)). Previous chromatin capture data demonstrated that SNP rs11154337 interacts with canonical promoters in human umbilical vein endothelial cells ( Fig.15A(AD)), providing a putative explanation for the long-range regulation of SNP rs11154337 and the canonical promoter of NCOA7. Consistent with the observed differences in NCOA7 expression and our previous findings with NCOA7 knockdown, lysosomal activity, sterol homeostasis, and immune activation in iPSC-ECs were allele-dependent. iPSC-ECs carrying the G allele (and thus higher NCOA7 expression) exhibited a concomitant increase in its binding partner ATP6V1B2, followed by a decrease in lysosomal pH, as evidenced by attenuated cleavage of SiR-lysosomes ( Fig. 12A (GI)). Furthermore, the presence of the G allele prevented lysosomal hypertrophy compared to iPSC-EC lines homozygous for the C allele, suggesting that proper lysosomal acidification and resulting sterol homeostasis are maintained ( Fig. 12B (J) and Fig. 15B (I,)).

[0256] Similar to RNAi targeting NCOA7, lower NCOA7 expression in homozygous C allele lines resulted in higher sterol content, which in turn drove higher expression of CH25H and its downstream metabolite 25HC (the enzyme responsible for generating downstream oxidative species such as 7HOCA) ( Fig. 12B (KO))——. With increased production of 7HOCA, homozygous C allele iPSC lines showed enhanced endothelial cell immune activity, as reflected by increased VCAM1 expression and immune cell adhesion ( Fig. 12B (PS)). Thus, consistent with the association of the G allele of SNP rs11154337 with protection against oxysterol production and PAH severity, the G allele increases NCOA7 expression, its downstream regulation of lysosomal acidification, oxysterol production, and the resulting EC immune activation.

[0257] Structural modeling and molecular simulations to identify novel therapeutic activators of NCOA7: To identify small molecule activators of NCOA7, we performed structure-based computations consisting of three parts: druggability simulations, pharmacophore modeling, and virtual screening ( Fig.16A (AC). Drugability simulations were performed using the model structure of NCOA7 in the presence of explicit water and probe molecules representing drug-like fragments. Six independent runs of 40 ns each were performed using the probe molecules acetamide, acetate, benzene, imidazole, isobutane, isopropanol, and isopropylamine. In three of the runs, the molecular pocket was distinguished by its high affinity for binding the probe molecules ( Fig.16A (A), cyan spheres). The site also shows the hinge residues from Gaussian network model (GNM) analysis of NCOA7 ( Fig.17 (A)). The hinge residues at or near the binding pocket are L83 (model 1), L72 (model 2), and E66 and W81 (model 3) ( Fig.17 (B)). Of note, previous studies have shown that hinge sites play a key role in mediating the functional dynamics of proteins and have therefore been used as target sites for binding small molecule modulators of protein function. Therefore, the identified molecular pockets were selected for further analysis using pharmacophore modeling enhanced by druggability simulations and GNM analysis.

[0258] Using our tool Pharmmaker, we performed pharmacophore modeling and subsequently selected high-affinity residues at the identified molecular pockets (i.e., N62, E66, P80, and W81; Fig.16A (B))(JYLee, 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. It is worth noting that in the probe molecules, two benzenes showed a high tendency to interact with N62, P80 and W81, while an isopropylamine interacted with E66 ( Fig.16A (B), benzene in black and isopropylamine in blue). Molecular dynamics (MD) snapshots showing multiple commonly observed (i.e., entropically favorable) interactions simultaneously were used to construct a pharmacophore model consisting of a hydrogen bond donor, three hydrophobic rings, and two aromatic rings ( Fig.16A (C)).

[0259] Then, the pharmacophore model was screened against ZINC (T. Sterling, JJ Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)) and MolPort small molecule libraries by Pharmit (T. Sterling, JJ Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)) server to obtain a set of compounds. The highest-scoring compounds were selected as targets for further experimental validation. Compound MolPort-004-267-958 (referred to herein as 958) produced preliminary data indicating that it is an activator of NCOA7 relative to other predicted compounds. Then, we attempted to further examine the binding behavior of 958 by MD simulation.

[0260] Optimization of activator 958 leads to analogs 958 with stronger binding affinity ami : To generate molecules that bind more tightly to the NCOA7 molecular pocket, we performed MD simulations. Therefore, all-atom 0.6 μs MD simulations of 958 (or three separate 0.2 μs runs) were performed to characterize the most critical functional groups and interactions. Based on these simulations, we designed analogs of 958 in which the O 15 Atoms are N 15 -H replacement to generate an amide functional group, the resulting compound is referred to herein as 958 ami ( Fig.16A (D, E)). Then use 958 ami The MD simulations performed further elucidated its enhanced activity. To compare the structures of the parent and analogs, we selected nine residues with high interaction affinity with the compounds: H56, N62, I65, E66, A69, R70, Q77, G78, and W81. The total contact duration of these residues was greater than 0.35 μs, while that with 958 or 958 ami The contact duration is 0.6μs. Fig.18 (AC)). Strong interactions (defined as contact duration greater than 0.3 μs) are caused by H56-O1 in 958 relative to 958 ami N62-O 10 、E66-O 10 / O 11 and W81-N 15 / O 17 form( Fig.16A (D); solid orange line). Notably, atoms 16 to 34 do not make any significant interactions with the NCOA7 binding pocket; however, 15 -H replaces O 15 This results in multiple strong interactions with many residues in the pocket. 15 The substitutions exhibit strong interactions with I65 and W81, induce new interactions between atoms 16 to 34 and A69, R70, and Q77, and strengthen the C 23 / C 24 -G78 and O 10 / O 11 -N62 / E66 interaction. Finally, hydrogen bond analysis was used to analyze the interaction between donor and acceptor atoms. The cutoff distance and 160° angle identify E66 and 958 ami There is a significantly higher propensity to form hydrogen bonds.

[0261] The binding affinity was calculated using PRODIGY-LIG (T. Sterling, JJ Irwin, ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337 (2015)), indicating that 958 ami Binding to NCOA7 is more stable than the parent compound 958 ( Fig.19 (A)). The corresponding binding pose at 100 to 200 ns has a binding affinity of -8.16 ± 0.16 kcal / mol ( Fig.16A (E), Run 1). 958 ami There are two other stable postures: one is that W81 loses the stability with N 15 The interaction ( Fig.19 (B), run 2, -7.71 ± 0.16 kcal / mol), and another in which the compound is rotated upside down in the pocket ( Fig.19 (B), run 3, -8.83 ± 0.19 kcal / mol).

[0262] Administration of NCOA7 activator 958 ami Disease reversal in PAH models: To evaluate the 958 ami To investigate the downstream molecular functions of NCOA7, we performed a proximity ligation assay to assess the ATP6V1B2-NCOA7 interaction. ami Significant induction of the number of expansions per cell, indicating an enhancement of molecules at the lysosomal level ( Fig. 16B (F, G)). Use 958 ami Maintaining lysosomal acidification also prevented the induction of CH25H in the presence of IL-1β, which further corresponded to a reduction in EC immunoreactivity, as indicated by VCAM1 expression and immune cell adhesion to the monolayer ( Fig. 16B (HL)).

[0263] Next, we tried to confirm 958 ami To investigate whether endothelium is protected from immune activation in the proinflammatory monocrotaline PAH rat model. After monocrotaline loading, rats were injected intraperitoneally with DMSO or 958 ami (7.5 mg / kg), up to 10 days ( Fig. 16B (M)). Compared with the vehicle control, 958 ami Treated rodents showed neither significant hepatotoxicity nor renal toxicity nor alterations in left ventricular function ( Fig. 20 (BK) ). NCOA7 activator 958 amiTreated rats showed decreased CH25H expression, as well as VCAM1 expression in the endothelium and CD11b in the pulmonary vasculature + The corresponding decrease in mononuclear cell infiltration ( Fig. 16B and 16C (NQ)). As a result, the pulmonary vasculature showed reduced muscularization, which corresponded to a significant reduction in right ventricular hypertrophy and RVSP ( Fig. 16C (RT)). Overall, these data identified 958 ami As a new therapeutic agent to reverse the pathogenesis of PAH, it has potential significance related to immune disorders.

[0264] By utilizing large-scale, multidimensional genomic and metabolomic analyses and accompanying mechanistic experiments, we found that NCOA7 regulates lysosomal activity and EC sterol metabolism to function as a homeostatic brake and prevent oxysterol-induced inflammation, EC dysfunction, and PAH. Most notably, the presence of the G allele at SNP rs11154337 resulted in enhanced NCOA7 expression, which reduced inflammation in PAH and established mechanistic evidence for a potential genetic association between SNP rs11154337 and PAH mortality, as well as a metabolomic association between the oxysterol signature and PAH severity. Ultimately, this work establishes a new paradigm linking basic lysosomal biology and oxysterol metabolism to EC behavior, with broad implications for the development of molecular diagnostics and treatments for PAH as well as other inflammatory vascular disorders.

[0265] The identification of NCOA7 as a master controller of PAH has broad implications for human disease. The role of NCOA7 described herein in immune regulation is thought to point to a broader role for NCOA7 isoforms and related proteins. Although recent studies have reported unique activities of the short-length isoforms of NCOA7, the findings described herein indicate that both isoforms have additive or synergistic behaviors in regulating oxysterol-mediated EC inflammation. NCOA7 isoforms carry the Tre2 / Bub2 / Cdc16 (TBC), lysin motif (LysM), and domain catalytic (TLDc) domains. All TLDc-containing proteins physically interact with the V-ATPase, defining a new class of V-ATPase regulatory proteins.

[0266] At the cellular level, this work emphasizes the extensive role of lysosomes in EC function and PAH. Previous clinical observations have shown the relationship between rare, recessive, loss-of-function lysosomal storage diseases and pulmonary vascular disease. For example, mutations in various V-ATPase subunits (e.g., ATP6V1A and ATP6V1E1) in humans can manifest as pulmonary artery stenosis or hypoplasia and right ventricular hypertrophy (T. Van Damme et al., Mutations in ATP6V1E1 or ATP6V1A CauseAutosomal-Recessive Cutis Laxa. Am J Hum Genet 100, 216-227 (2017)). PAH has been found in mucolipidosis (a disease caused by lysosomal enzyme processing dysfunction). Patients with Gaucher disease (a condition caused by lysosomal β-glucosidase deficiency and known to be associated with grade V PH) have been reported to have high pulmonary artery pressure ( Figure 6B (F, G). In addition, Niemann-Pick disease and Fabry disease present with severe lung dysfunction, which often co-occurs with grade III PH. In support of these rare genetic diseases, the association between the homozygous C / C genotype and worse survival in PAH provides broader and more definitive evidence for a causal link between lysosomal dysfunction and PAH. In fact, guided by the average C allele frequency of SNPrs1115447 in the global population (~0.48 to 0.52), approximately one-quarter of PAH patients carrying the C / C genotype would be expected to face worse mortality. In addition, based on the principle of cooperative heterozygosity previously reported in the context of BMPR2 mutations in familial PAH, it remains to be seen whether monoallelic carriers of known familial PAH mutations or lysosomal enzyme mutations are more likely to present with PAH or other lysosomal storage diseases if accompanied by the SNP rs1115447 C / C genotype.

[0267] In conclusion, through multidimensional analysis of genomic and metabolomic datasets combined with in vitro and in vivo mechanistic validation, we identified essential and SNP-dependent roles for NCOA7 and its control of lysosomal activity and sterol homeostasis to moderate inflammation, EC dysfunction, and PAH. ami and its derivatives. In vitro and in vivo studies have shown that compound 958 ami Preventing oxysterol-mediated endothelial immune activation and reversing disease in monocrotaline rats with PAH, and therapies (such as 958 ami ) are a powerful complement to existing vasodilator therapies and other disease-modifying agents in development that target cell proliferation and survival pathways.

[0268] Example 2: In vitro virus assay

[0269] SNPs rs11154337 and 958 regulate the entry of various pseudotyped enveloped viruses. Fig.21 As shown, iPSCs transfected with human ACE2 receptor showed reduced entry of multiple pseudotyped coronaviruses and herpes viruses in the presence of the G allele at SNP rs11154337, which led to increased NCOA7 expression. In addition, the NCOA7 activator 958 was applied to enhance the activity of NCOA7 to prevent pseudotyped SARS-CoV-2 infection (D614G spike) in HEK293 cells transfected with human ACE2 receptor. 958 parent (2μM) and 958 ami (5 μM) reduced BA.2 spike pseudotyped virus infection in iPSCs transfected with human ACE2 receptor. In addition, 958 mother (2 μM) and 958 ami (5 μM) reduces D614G spike pseudotype virus infection in iPSCs transfected with human ACE2 receptor.

[0270] Example 3: In vivo SARS-CoV2 assay

[0271] refer to Fig. 22 , lung inflammation and mortality were reduced in coronavirus-infected human ACE2 transgenic mice treated with the NCOA7 activator 958. The mortality of human ACE2 transgenic mice infected with two different coronavirus strains was significantly improved when treated with 958. In addition, when treated with 958, mouse lung tissues showed reduced viral load and attenuation of various pro-inflammatory markers (IL-1α, IL-1β, IFN-γ, VCAM1, and ICAM1) as detected by RT-qPCR.

[0272] Example 4: Reducing lung inflammation in bacterial infection in vivo

[0273] Mice infected with Klebsiella pneumoniae. Fig.23 These mice showed amelioration of acute lung injury when treated with the NCOA7 activator 958. Fig.23 (A, B) Representative images of lung tissue sections prepared from H&E staining of mice treated with DMSO (5 mg / kg IP daily for 3 days) 48 hours after intratracheal infection with Klebsiella pneumoniae. The mice treated with 958 showed reduced inflammation ( Fig.23 (C to E)), as shown by ELISA of lung homogenates for the proinflammatory cytokines IL-1β, TNF-α, and IL-6.

[0274] Example 5 - Stroke

[0275] NCOA7 deficiency worsens survival in a mouse model of ischemic stroke. Fig.24 Wild-type mice and Ncoa7-deficient mice were used for the experimental model of ischemic stroke. The mice underwent sham surgery or transient middle cerebral artery occlusion (tMCAO) for 60 minutes before restoration of cerebral blood flow. Then, the mice were collected at 24 and 48 hours for further studies. Compared with wild-type controls, a significantly reduced survival rate was observed in Ncoa7 knockout mice at 24 and 48 hours after tMCAO. There was no significant change in body weight 24 or 48 hours after tMCAO. Using laser Doppler flowmetry, the regional cerebral blood flow (rCBF) of the contralateral (CL) and ipsilateral (IL) cerebral cortex of the mice was quantified. Compared with wild-type controls, the rCBF in the IL cerebral cortex of Ncoa7-deficient mice was increased at 24 hours after tMCAO.

[0276] NCOA7 deficiency increases infarct volume and capillary leakage after ischemic stroke in mice. Fig.25 As shown, mouse brain tissue was cut into 30-micron sections before immunofluorescence staining. Brain sections were stained with the neuronal marker microtubule-associated protein 2 (MAP2) to assess infarct volume after tMCAO. In NCOA7-deficient mice, infarct volume was significantly increased after ischemic stroke, indicating more neuronal cell death. There was no significant change in tissue swelling compared with wild-type controls. To assess capillary leakage, brain sections were stained for the plasma protein albumin. It is noteworthy that NCOA7-deficient mice showed a more obvious leakage trend of albumin into brain tissue after tMCAO, indicating that brain microvascular damage was more severe compared with wild-type controls.

[0277] NCOA7 deficiency leads to exacerbated neuroinflammation after ischemic stroke. Fig.26 As shown, mouse brain sections were stained using an immunofluorescence protocol. The brain was stained with astrocyte marker glial fibrillary acidic protein (GFAP), microglia marker ionized calcium binding adapter molecule 1 (IBA1) and nuclear marker (DAPI). Images in the cortex (Ctx) and striatum (Str) of wild-type and Ncoa7 knockout mice after tMCAO were acquired using a confocal microscope. Neuroinflammation in NCOA7-deficient mice was significantly worsened, as shown by swelling of astrocyte processes in cortical and striatal tissues. In addition, microglia in knockout mice were hypertrophic and elongated compared to wild-type controls. These data indicate that there is a large amount of reactive gliosis and neuroinflammation in NCOA7-deficient brains after tMCAO.

[0278] NCOA7 deficiency leads to significant hypomyelination in a mouse model of ischemic stroke. Fig. 27 Brain sections were stained with myelin basic protein (MBP) using an immunofluorescence protocol. Global myelination was significantly enhanced in NCOA7-deficient mice, especially at the level of the corpus callosum (CC) and external capsule (EC). Notably, there was no significant difference in the contralateral (CL) hemisphere compared to the ipsilateral (IL) hemisphere after tMCAO.

[0279] The present invention has been described with reference to certain exemplary embodiments. However, those skilled in the art will recognize that various substitutions, modifications, or combinations may be made to any of the exemplary embodiments without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the description of the exemplary embodiments, but rather by the originally filed claims.

Claims

1. A method of treating inflammation in a patient, such as a human patient, comprising: An amount of a compound having the following structure is administered to the patient in an amount effective to reduce inflammation in the patient: Wherein R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, and X3 are independently N or C; X4 is located at the ortho, meta or para position to X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H; -C 1-3 Alkyl; halogen; -NO2; -CN; -CF3; -SO2R4, where R4 is -OH or -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 1-3 Alkyl; -OR8, wherein R8 is -H or -C 1-3 Alkyl; -OC(O)-R9, wherein R9 is -H or -C 1-3 Alkyl; -C(O)-R 10 , where R 10 -H or -C 1-3 Alkyl; or -C(O)-R 11 -R 12 , where R 11 is O or NH and R 12 -H or -C 1-3 alkyl; 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) ; MolPort-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 ZINC9583892, or a pharmaceutically acceptable salt thereof.

2. The method of claim 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, neurodegenerative disease, cancer, autoimmune disease, or innate and acquired immune diseases.

3. The method of claim 1, wherein the patient suffers from vascular inflammation.

4. The method of claim 1, wherein the inflammation is associated with a viral infection or a bacterial infection.

5. The method of claim 2, wherein the inflammation is associated with a coronavirus infection in the patient, optionally associated with severe acute respiratory syndrome caused by a coronavirus infection in the patient.

6. The method of claim 2, wherein the inflammation is associated with a bacterial infection in the patient.

7. The method of claim 6, wherein the bacterial infection is a Klebsiella pneumoniae infection in the patient, optionally reducing or preventing lung damage in the patient.

8. The method of claim 1, wherein the inflammation is associated with one or more of: pulmonary arterial hypertension (PAH), pulmonary hypertension caused by left heart disease, pulmonary hypertension caused by lung disease, pulmonary hypertension caused by chronic thrombosis in the lungs, and pulmonary hypertension of unknown etiology.

9. A method of treating pulmonary arterial hypertension (PAH) in a patient, comprising administering to the patient a compound having the structure: Wherein R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, and X3 are independently N or C; X4 is located at the ortho, meta or para position to X1 and is N or C; Y2 is -H or -C 1-3 Alkyl, halogen or -NO2; Y1 is -H; -C 1-3 Alkyl; halogen; -NO2; -CN; -CF3; -SO2R4, where R4 is -OH or -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 1-3 Alkyl; -OR8, wherein R8 is -H or -C 1-3 Alkyl, C 1-3 Alkoxy; -OC(O)-R9, wherein R9 is -H or -C 1-3 Alkyl, ester; -C(O)-R 10 , where R 10 -H or -C 1-3 Alkyl; or -C(O)-R 11 -R 12 , where R 11 is O or NH and R 12 -H or -C 1-3 alkyl; 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) ; MolPort-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 ZINC9583892, or a pharmaceutically acceptable salt thereof.

10. The method according to any one of claims 1 to 9, wherein Z is NH.

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

12. The method according to any one of claims 1 to 10, wherein R1 and R2 are H.

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

14. The method according to any one of claims 1 to 9, wherein the compound is compound 958 ami , which has the following exemplary structure: or a pharmaceutically acceptable salt thereof.

15. The method of any one of claims 1-14, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient in an amount effective to reduce inflammation in the patient or to treat pulmonary hypertension in the patient.

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

17. according to the method described in any one of claims 1-16, wherein said patient is heterozygous or homozygous for C at rs11154337.

18. according to the method described in any one of claims 1-16, wherein the patient is homozygous for C at rs11154337.

19. Method 17 or 18, further comprising obtaining genetic data of the patient and determining whether the patient has one or two alleles of C at rs11154337.

20. The method of claim 17 or 18, further comprising determining whether the patient has one or two alleles that are C at rs11154337.

21. The method of claim 5, wherein the coronavirus infection is one or more of: Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), or a disease caused thereby, such as Coronavirus Disease 2019 (COVID-19).

22. The method of claim 21, wherein the coronavirus is SARS-CoV-2.

23. The method of claim 1, for reducing the infectivity of a coronavirus or herpes virus infection in a cell.

24. A compound comprising the following structure: in R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, and X3 are independently N or C; X4 is located at the ortho, meta or para position to X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H; -C 1-3 Alkyl; halogen; -NO2; -CN; -CF3; -SO2R4, where 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 1-3 Alkyl; -OR8, wherein R8 is -H or -C 1-3 Alkyl; -OC(O)-R9, wherein R9 is -H or -C 1-3 Alkyl; -C(O)-R 10 , where R 10 -H or -C 1-3 Alkyl; or -C(O)-R 11 -R 12 , where R 11 is O or NH and R 12 -H; -C 1-3 alkyl, or a pharmaceutically acceptable salt thereof, excluding MolPort-004-267-958.

25. The compound of claim 24, wherein Z is NH.

26. The compound according to claim 24 or 25, wherein R1 and R2 are independently Me or H.

27. The compound of claim 24 or 25, wherein R1 and R2 are H.

28. A compound according to claim 24 having the following structure: or a pharmaceutically acceptable salt thereof.

29. A pharmaceutical composition comprising: A compound, which either: Has the following structure: Wherein R1 and R2 are independently -H or -C 1-3 alkyl; Z is O or NH; X1, X2, and X3 are independently N or C; X4 is located at the ortho, meta or para position to X1 and is N or C; Y2 is -H, -C 1-3 Alkyl, halogen or -NO2; Y1 is -H; -C 1-3 Alkyl; halogen; -NO2; -CN; -CF3; -SO2R4, where R4 is -OH or -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 1-3 Alkyl; -OR8, wherein R8 is -H or -C 1-3 Alkyl; -OC(O)-R9, wherein R9 is -H or -C 1-3 Alkyl; -C(O)-R 10 , where R 10 -H or -C 1-3 Alkyl; or -C(O)-R 11 -R 12 , where R 11 is O or NH and R 12 -H or -C 1-3 Alkyl; or a pharmaceutically acceptable salt thereof; Or the compound is selected from one or more of the following: 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 (ZINC9 or a pharmaceutically acceptable salt thereof; and Pharmaceutically effective excipients, The composition comprises an amount of the compound effective to treat or reduce inflammation, cardiovascular inflammation, vascular inflammation (e.g., diseases with vascular endothelial inflammation or with 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 in a patient, such as a human patient, or for treating a coronavirus infection, such as a SARS-CoV-2 infection, in a patient, such as a human patient.

30. The composition of claim 29, wherein Z is NH.

31. A composition according to claim 29 or 30, wherein R1 and R2 are independently Me or H.

32. The composition of claim 29 or 30, wherein R1 and R2 are H.

33. The composition according to claim 29, having the following structure: or a pharmaceutically acceptable salt thereof.

34. The composition of claim 29, wherein the compound is MolPort-004-267-958.

35. A method for treating a patient, wherein the patient has a C at SNP rs11154337 and suffers from inflammation and / or an inflammatory disease, such as cardiovascular inflammation or vascular inflammation; or the inflammation and / or inflammatory disease 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, neurodegenerative disease, cancer, autoimmune disease, or innate and acquired immune disease, The method comprises using gene editing, such as but not limited to CRISPR / Cas9 or TALEN-based methods, to change one or more Cs at SNPrs11154337 to Gs.

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