WO2023208199A1

WO2023208199A1 - USE OF ACTIVE MOLECULES FOR REGULATING HIF-3α IN TREATMENT OF DISEASES

Info

Publication number: WO2023208199A1
Application number: PCT/CN2023/091625
Authority: WO
Inventors: 武大雷; 宋淳; 张成城; 庄静静; 刁晓彤
Original assignee: 山东大学; 济南成城生物技术有限公司
Priority date: 2022-04-29
Filing date: 2023-04-28
Publication date: 2023-11-02
Also published as: CN116407635A

Abstract

The use of active molecules for regulating HIF-3α in preparation of drugs for treating diseases. The diseases are selected from infections and tumors, and one active molecule is oleoylethanolamide. A pharmaceutical composition for treating infections or tumors, comprising: an active ingredient (a), being a therapeutically effective amount of an anti-infection or antineoplastic drug; and/or an active ingredient (b), being a therapeutically effective amount of the active molecule for regulating HIF-3α; and optionally, a drug carrier.

Description

Application of active molecules that regulate HIF-3α in treating diseases

This application is based on the application with CN application number 202210467109.0 and the filing date is April 29, 2022, and claims its priority. The disclosure content of the CN application is hereby incorporated into this application as a whole.

Technical field

This application belongs to the field of biomedicine, and specifically relates to the use of active molecules that regulate HIF-3α and pharmaceutical compositions thereof in the preparation of drugs for the treatment of diseases (such as infections or tumors).

Background technique

Viral infections remain a major threat to global public health. Although viral treatments and vaccinations have greatly reduced the impact of viral infectious diseases over the past few decades, with the emergence of new pathogenic microorganisms, the increase in drug-resistant pathogenic microorganisms, and the increase in immunosuppressed hosts, viral Infection morbidity and mortality remain high.

Hypoxia-inducible factor (HIF) is a type of transcription factor with very important functions in the human body. It can help the body or cells adapt to a hypoxic environment by regulating the transcription of genes related to erythropoiesis, angiogenesis, and anaerobic metabolism. . Therefore, abnormal activity of the HIF pathway is closely related to various diseases such as cancer and anemia. The transcriptionally active HIF protein complex consists of two subunits, HIF-α and ARNT (aryl hydrocarbon receptor nuclear translocator, also known as HIF-1β), and HIF-α contains three subtypes (HIF-1α, 2α and 3α).

Compared with HIF-1α and HIF-2α, HIF-3α is the HIF-α subtype with the least understood structure and function and the least conserved structure and function. One of the main reasons is that the gene encoding HIF-3α can produce a variety of mRNAs of different lengths by changing the transcription start site and alternative splicing (at least 3 types in mice and 7 types in humans have been found). Corresponds to proteins of different sizes, so the functions are relatively complex and diverse. Simply put, the longer HIF-3α spliceosome has a similar domain composition to the other two isoforms and can form a stable dimer with ARNT, but its C terminus only contains a transcriptional activation region (HIF-1α and HIF -2α each has two), so its transcriptional activity is weak. The shorter HIF-3α spliceosome is even more unique, especially IPAS (inhibitory PAS) and HIF-3α4, which only contain about half the length of the N-terminus. They can directly bind to HIF-1α or HIF-2α and inhibit the transcriptional activity of both. , but the specific molecular mechanism of how it is combined is currently unclear.

Therefore, it is of great significance to find small molecules that can bind HIF-3α and use them as tool molecules to study the function of HIF-3α, and then develop drugs targeting HIF-3α.

Contents of the invention

After experimental verification, it was found that OEA is an endogenous agonist of HIF-3α and can activate the transcription and translation of the HIF-3α downstream gene HSPA6. Overexpression of HIF-3α in Hep3B cells can significantly affect cellular antiviral resistance Related signaling pathways, such as the anti-measles virus signaling pathway, the anti-influenza A virus signaling pathway, the anti-hepatitis C virus signaling pathway, etc., have antiviral effects.

Therefore, one object of the present invention is to provide the use of molecules that regulate HIF-3α levels or pharmaceutical compositions containing molecules that regulate HIF-3α levels in the preparation of drugs for treating diseases.

In some embodiments, the molecule is a small chemical molecule or a biological macromolecule, such as siRNA, miRNA, antisense nucleic acid or PROTAC drug, etc.

In some embodiments, the molecule is an agonist of HIF-3α.

In some embodiments, the molecule is OEA (oleoylethanolamine):

In some embodiments, the molecule is an antagonist or inhibitor of HIF-3α.

In some embodiments, the disease is selected from infections and tumors.

In some embodiments, the infection is an infection caused by a virus (eg, a pathogenic virus).

In some embodiments, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae, and Herpesviridae.

In some embodiments, the Paramyxoviridae virus is measles virus.

In some embodiments, the Orthomyxoviridae virus is an influenza A virus (eg, H1N1).

In some embodiments, the Flaviviridae virus is Hepatitis C virus.

In some embodiments, the Herpesviridae virus is a herpesvirus or Epstein-Barr virus.

In some embodiments, the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, melanoma, glioma, or sarcoma.

In another aspect, the present invention provides a pharmaceutical composition for treating viral infection, which includes: active ingredient (a), a therapeutically effective amount of an antiviral drug; and/or active ingredient (b), a therapeutically effective amount of an antiviral drug An active molecule that modulates HIF-3α; and optionally a pharmaceutical carrier.

In some embodiments, active ingredients (a) and (b) are in the same or different formulation units.

In some embodiments, the antiviral drug is selected from amantadine, rimantadine, enfuvirtide, maraviroc, acyclovir, ganciclovir, valacyclovir, famciclovir, foscarnet , lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, efavirenz, nevirapine, saquinavir, oseltamivir, zanamivir, riba Welin or interferon, etc.

In some embodiments, the active molecule that regulates HIF-3α is OEA or the like.

In some embodiments, the infection is a viral infection, such as an infection caused by a pathogenic virus. In some implementations In this case, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Herpesviridae. In some embodiments, the Paramyxoviridae virus is measles virus. In some embodiments, the Orthomyxoviridae virus is an influenza A virus (eg, H1N1). In some embodiments, the Flaviviridae virus is Hepatitis C virus. In some embodiments, the Herpesviridae virus is a herpesvirus or Epstein-Barr virus.

In some embodiments, the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, melanoma, glioma, and sarcoma.

In another aspect, the present invention provides the use of OEA in the preparation of HIF-3α agonists.

In another aspect, the present invention provides the use of a molecule that modulates HIF-3α levels or a pharmaceutical composition containing a molecule that modulates HIF-3α levels for treating a disease.

In some embodiments, the molecule is a small chemical molecule or a biological macromolecule, for example, selected from the group consisting of siRNA, miRNA, antisense nucleic acids, and PROTAC drugs.

In some embodiments, the molecule is an agonist of HIF-3α.

In some embodiments, the molecule is OEA (oleoylethanolamine):

In some embodiments, the molecule is an antagonist or inhibitor of HIF-3α.

In some embodiments, the disease is selected from infections and tumors.

In some embodiments, the infection is a viral infection, such as an infection caused by a pathogenic virus. In some embodiments, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae, and Herpesviridae. In some embodiments, the Paramyxoviridae virus is measles virus. In some embodiments, the Orthomyxoviridae virus is an influenza A virus (eg, H1N1). In some embodiments, the Flaviviridae virus is Hepatitis C virus. In some embodiments, the Herpesviridae virus is a herpesvirus or Epstein-Barr virus.

In another aspect, the present invention provides an anti-infection or anti-tumor method, which includes administering to a subject in need thereof an effective amount of a molecule that modulates HIF-3α levels, a medicament containing a molecule that modulates HIF-3α levels. composition or any of the steps of the pharmaceutical composition described above.

In some embodiments, the molecule is an agonist of HIF-3α.

In some embodiments, the molecule is OEA (oleoylethanolamine):

In some embodiments, the molecule is an antagonist or inhibitor of HIF-3α.

In some embodiments, active ingredient (a) and active ingredient (b) in the pharmaceutical composition are administered to the subject simultaneously, sequentially or sequentially.

In some embodiments, the disease is selected from infections and tumors.

In another aspect, the present invention provides an anti-infection or anti-tumor drug screening method, which includes the following steps:

(1) Provide test molecules, HIF-3α (especially HIF-3αPAS-B) and HIF-3α overexpressing cells;

(2) Determine the binding between the test molecule and HIF-3α. If the two are combined, proceed to the next step;

(3) Treat the cells with the test molecule, measure the transcription level of HIF-3α downstream genes (such as HSPA6) in the cells, and compare with the negative control group; when the expression of the HIF-3α downstream genes is higher than In the case of a negative control group, the test molecule is determined to be an anti-infection or anti-tumor drug candidate.

definition

Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technology as used herein are intended to mean technology as commonly understood in the art, including those variations or equivalent technology that would be apparent to those skilled in the art. Moreover, the laboratory procedures used in this article such as genomics, nucleic acid chemistry, and molecular biology are routine procedures widely used in the corresponding fields. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

In the present invention, the virus is a tiny, submicroscopic particle without a complete cell structure that can be replicated using a host cell system.

In the present invention, the antiviral drugs refer to drugs that can directly inhibit or kill viruses, interfere with virus adsorption, prevent viruses from penetrating cells, inhibit virus biosynthesis, inhibit virus release, or enhance the host's antiviral ability.

In the present invention, the pharmaceutical composition may further comprise a pharmaceutical carrier. The pharmaceutical carrier is commonly used in this field.

In the present invention, OEA is taken as an example to reveal the use of active molecules that regulate HIF-3α levels in the preparation of antiviral drugs. The active molecules include but are not limited to siRNA, miRNA, antisense nucleic acid, PROTAC drugs and other forms. Viruses include, but are not limited to, measles virus, influenza A virus, hepatitis C virus, herpes virus, Epstein-Barr virus and other pathogenic viruses.

The active molecule or pharmaceutical composition for regulating HIF-3α levels according to the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, and elixirs. tablets, tablets, suppositories, injections (including injections, sterile powder for injection and concentrated solution for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage. The preferred dosage form is injection. This injection may be a sterile injectable solution. For example, sterile injectable solutions can be prepared by incorporating the necessary dose of the antibody of the invention into a suitable solvent, and optionally adding other desired ingredients (including but not limited to pH adjusters, surfactants, adjuvants , ionic strength enhancer, etc., penetrant, preservative, diluent or any combination thereof), and then filtered and sterilized. In addition, for convenience of storage and use, sterile injectable solutions can be prepared as sterile lyophilized powder (for example, by vacuum drying or freeze-drying). This sterile lyophilized powder can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use. The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania : Mack Publishing Company, 1995), and include but are not limited to: Ph adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, Ph adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.

As used herein, the term "effective amount" may refer to an amount effective at the dose and period of time required to achieve the desired effect. The effective amount may vary depending on factors such as the type of disease or condition being treated, the anatomy of the particular target organ being administered, the size of the patient, or the severity of the disease or symptoms. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation.

The inventors verified that OEA is an endogenous ligand of HIF-3α at the molecular level through SPR method, and its KD value is 14.0 μM. Overexpression of HIF-3α in HEK293 cells and administration of OEA can significantly activate the transcription and translation of the HIF-3α downstream gene HSPA6. In addition, transcriptomic KEGG analysis of overexpressing HIF-3α in Hep3B cells found that overexpression of HIF-3α can significantly affect cellular antiviral-related signaling pathways, such as anti-measles virus signaling pathways, anti-influenza A virus signaling pathways, and anti-influenza virus signaling pathways. Hepatitis C virus signaling pathway, etc., play an antiviral role. Overexpression of HIF-3α in Hep3B cells and administration of OEA can significantly exert anti-influenza A virus H1N1 effects.

The human liver cancer cell line Hep3B and the human embryonic kidney cell HEK293 used in the present invention are commonly used cell lines in the fields of scientific research and drug development.

Description of the drawings

The drawings described here are used to provide a further understanding of the present invention and constitute a part of this application. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 shows SPR detection of the binding ability of OEA to HIF-3αPAS-B.

Figure 2 shows that OEA activates the transcription of HIF3-α downstream target gene HSPA6.

Figure 3 shows transcriptomic KEGG analysis of Hep3B cells overexpressing HIF-3α.

Figure 4 shows that activating the HIF-3α signaling pathway in Hep3B cells can protect against influenza A virus H1N1.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application or uses. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Example 1 Detection of the binding ability of OEA to HIF-3α by surface plasmon resonance (SPR)

Exogenously express HIF-3αPAS-B protein, and use CM5 amino-coupling chip to determine the binding ability of HIF-3αPAS-B domain and OEA.

1 Experimental materials and methods

1) Pre-enrichment

Dilute the protein to 0.67 mg/ml, take 3 μL and mix it with the sodium acetate buffer solution (pH is 5.5, 5.0, 4.5, 4.0 respectively). The final protein concentration is 20 μg/ml. Set the manual program according to Table 1 to make the protein sodium acetate The solution is pre-enriched sequentially from high pH to low pH. Running buffer: 10mM HEPES (pH 7.4), 400mM NaCl, 0.02% volume P-20.

Table 1 Pre-enrichment program settings

2) Amino coupling

Perform amino coupling according to the "Amine" method, and set the coupling amount for protein coupling. Couple MBP protein to channel 1 (flow cell 1) and MBP-HIF-3αPAS-B to channel 2 (flow cell 2). Running buffer: 10mM HEPES (pH 7.4), 400mM NaCl, 0.02% P-20.

3) Affinity determination

Load eight concentrations of OEA (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM, 25μM, 30μM) into channel 1 and channel 2 of the CM5 chip according to the setting procedure in Table 2. In order to reduce signal errors caused by small differences in DMSO concentration, it is necessary to use a running buffer with a DMSO concentration of 4.5 to 5.5% for solvent correction. The configuration of the solvent correction buffer solution is shown in Table 3. Running buffer: 10mM HEPES (pH 7.4), 400mM NaCl, 0.02% P-20 and 5% volume DMSO.

Table 2 Affinity determination program settings

2Experimental results

As shown in Figure 1, OEA can effectively bind to the HIF-3α PASB domain, with a K _D value of 14.0 μM.

Example 2 OEA activates the transcription of HIF-3α downstream gene HSPA6

1. Experimental materials and methods

1) HIF-3α transfected HEK 293 cells

A. When HEK 293 cells grow well and reach a density of 80%-90%, digest them with trypsin and spread them in a 12-well plate. When the cells adhere well and grow to a density of 50%-60%, transfect dye.

B. Transfection step: add 0.4 μg of DNA plasmid (control group: pCMV-Tag4 empty vector; experimental group: HIF-3α plasmid with pCMV-Tag4 vector) in 75 μl Mix well in buffer, then add 0.8μl of reagent, mix well, let stand at room temperature for 10 minutes, add dropwise to the 12-well plate, mix gently and continue to culture the cells for 24 hours.

2) RNA extraction experiment

Total RNA is extracted using the isopropyl alcohol precipitation method. The specific steps are as follows (taking a 12-well plate as an example):

A. After washing each well with 500μL PBS, add 500μL RNAiso plus and pipet several times. Transfer the solution to a 1.5mL EP tube.

B. Add 200 μL of chloroform into the 1.5 mL EP tube, mix it upside down for 30 seconds, let it stand at room temperature for 10 minutes, place it in a 4°C centrifuge, and centrifuge at 12,000 rpm for 15 minutes.

C. Transfer the supernatant to a new 1.5mL EP tube and add an equal volume of isopropanol to precipitate RNA. Mix upside down 10 times, let stand at room temperature for 10 minutes, place in a centrifuge at 4°C, and centrifuge at 12,000 rpm for 15 minutes.

D. Discard the supernatant, add 1 mL of 75% ethanol to resuspend the pellet, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the supernatant, open the cap of the EP tube, and evaporate the remaining liquid at room temperature. After 15-20 minutes, add an appropriate amount of DEPC water to dissolve the precipitate before proceeding to the next experiment.

3) Real-time quantitative fluorescence PCR experiment:

A. Reverse transcription: The concentration and A260/A280 ratio of the extracted RNA above are measured. After passing the test, each Adjust the concentration of group RNA to be consistent, and obtain cDNA by reverse transcription according to Table 4.

Table 4 Reverse transcription reaction conditions and system

B. Quantitative PCR reaction system (per well):

In this part, β-actin was selected as the internal reference gene, and the 2 ^-ΔΔCT method was used to calculate the relative expression of each gene. The q PCR experiment was performed according to the instructions of Shanghai Yisheng's SYBR Green kit, and three parallel experiments were designed for the detection of each gene. The reaction system is shown in Table 5, and the target gene primer sequence is shown in Table 6.

Table 5 q PCR reaction system and conditions

Table 6 Target gene primer sequence

2.Experimental results

As shown in Figure 2, HSPA6 is a downstream gene of HIF-3α. When HIF-3α is overexpressed, the expression of HSPA6 increases. When OEA (25μM, 24h) was added while overexpressing HIF-3α, the increase level of HSPA6 was higher than that of overexpressing HIF-3α alone. Therefore, OEA may be an endogenous agonist of HIF-3α and enhance the physiological function of HIF-3α.

Example 3 Overexpression of HIF-3α can activate antiviral signaling pathways in cells

1. Experimental materials and methods

1) HIF-3α transfected Hep3B cells

A. When Hep3B cells grow well and reach a density of 80%-90%, digest them with trypsin and spread them in a 12-well plate. When the cells adhere well and grow to a density of 50%-60%, transfection is performed. .

B. Transfection step: add 0.5 μg of DNA plasmid (control group: pCMV-Tag4 empty vector; experimental group: HIF-3α plasmid with pCMV-Tag4 vector) in 75 μl Mix well in buffer, then add 0.8μl of reagent, mix well, let stand at room temperature for 10 minutes, add dropwise to the 12-well plate, mix gently and continue to culture the cells for 24 hours.

2) RNA extraction experiment

D. Discard the supernatant and a white precipitate can be observed at the bottom of the tube. Add 1 mL of 75% ethanol to resuspend the pellet, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the supernatant, open the cap of the EP tube, and evaporate the remaining liquid at room temperature. After 15-20 minutes, add an appropriate amount of DEPC water to dissolve the precipitate and send it to Wuhan Boyue Zhihe Company for transcriptomic sequencing.

2.Experimental results

KEGG analysis of transcriptomics overexpressing HIF-3α in Hep3B cells showed (Figure 3) that HIF-3α can significantly affect cellular antiviral-related signaling pathways, such as anti-measles virus signaling pathways, anti-influenza A virus signaling pathways, and anti-influenza virus signaling pathways. Hepatitis C virus signaling pathway, etc.

Example 4 Activating the HIF-3α signaling pathway has anti-influenza A virus H1N1 activity

1. Experimental materials and methods

1)Virus virulence detection

Hep3B cells were plated and cultured in a 96-well plate, the medium was discarded, and the cells were washed twice with PBS. Use virus growth solution to dilute the virus 10 times to different concentrations, and add 100 μl to each well of a 96-well plate, with 8 duplicate wells for each concentration. Set up a normal control group, put the cells into culture adsorption for 2 hours, discard the virus solution, add 100 μl of virus growth solution, continue to culture, and observe the cell lesions until the cells no longer show new lesions. Observe and record the number of wells with cytopathic effects at each dilution ratio under a microscope, record the cytopathic effect (CPE), and calculate the virus' tissue cell half-infection dose (TCID ₅₀ ) according to the Reed-Muench method.

2) HIF-3α transfected Hep3B cells

A. When Hep3B cells grow well and reach a density of 80%-90%, digest them with trypsin and spread them in a 96-well plate. When the cells adhere well and grow to a density of 50%-60%, transfection is performed. .

B. Transfection step: add 60ng of DNA plasmid (pCMV-Tag4 empty or pCMV-Tag4 HIF-3α plasmid) in 10 μl Mix well in buffer, then add 0.1μl of reagent, mix well, let it stand at room temperature for 10 minutes, add it to a 96-well plate, mix gently and continue to culture the cells for 24 hours.

3) Cytopathic effect inhibition experiment (CPE)

After the cells were transfected and cultured for 24 hours, TCID ₅₀ virus and different compounds were added. The final concentration of DMSO was 0.1% and cultured for 48 hours. Cell viability was detected using CCK-8 method. Add 10 μl CCK-8 solution to each well, incubate at 37°C for 0.5-4 hours, and detect the absorbance at 450 nm.

2.Experimental results

In Hep3B cells, overexpression of HIF-3α can effectively inhibit H1N1 replication; on the basis of overexpression of HIF-3α, administration of the HIF-3α agonist OEA (25 μM) improved the antiviral activity, and combined with oseltamivir (1 μM) The activity was similar (Fig. 4).

The above embodiments are only exemplary embodiments of the present invention and are not used to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions should also be deemed to fall within the protection scope of the present invention.

Claims

Use of molecules that regulate HIF-3α levels or pharmaceutical compositions containing molecules that regulate HIF-3α levels in the preparation of drugs for treating diseases.
The use of claim 1, wherein the molecule is a chemical small molecule or a biological macromolecule, for example, selected from the group consisting of siRNA, miRNA, antisense nucleic acids and PROTAC drugs.
The use of claim 1 or 2, wherein the molecule is an agonist of HIF-3α;

Preferably, the molecule is OEA (oleoylethanolamine):
The use of claim 1 or 2, wherein the molecule is an antagonist or inhibitor of HIF-3α.
The use of any one of claims 1-4, wherein the disease is selected from infection and tumor.
The use of claim 5, wherein the infection is a viral infection, such as an infection caused by a pathogenic virus;

Preferably, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Herpesviridae;

Preferably, the Paramyxoviridae virus is measles virus;

Preferably, the Orthomyxoviridae virus is influenza A virus (such as H1N1);

Preferably, the Flaviviridae virus is hepatitis C virus;

Preferably, the Herpesviridae virus is a herpes virus or Epstein-Barr virus.
The use of claim 5, wherein the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, melanoma, glioma and sarcoma.
A pharmaceutical composition for treating infections or tumors, which contains: active ingredient (a), a therapeutically effective amount of an anti-infection or anti-tumor drug; and/or active ingredient (b), a therapeutically effective amount of regulated HIF-3α The active molecule; and optional pharmaceutical carrier;

Preferably, active ingredients (a) and (b) are in the same or different formulation units.
The pharmaceutical composition according to claim 8, wherein the anti-infective drug is an anti-viral drug. Preferably, the anti-viral drug is selected from the group consisting of amantadine, rimantadine, enfuvirtide, maraviroc, alfa. Aciclovir, ganciclovir, valacyclovir, famciclovir, foscarnet, lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, efavirenz, nevirapine , saquinavir, oseltamivir, zanamivir, ribavirin or interferon;

Preferably, the active molecule that regulates HIF-3α is OEA;

Preferably, the infection is a viral infection, such as an infection caused by a pathogenic virus; preferably, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Herpesviridae ; Preferably, the Paramyxoviridae virus is measles virus; Preferably, the Orthomyxoviridae virus is influenza A virus (such as H1N1); Preferably, the Flaviviridae virus is hepatitis C virus; Preferably, the Flaviviridae virus is hepatitis C virus; , the herpesviridae virus is herpes virus or Epstein-Barr virus;

Preferably, the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, melanoma, glioma and sarcoma.
Use of OEA in the preparation of HIF-3α agonists.
Molecules that regulate HIF-3α levels or pharmaceutical compositions containing molecules that regulate HIF-3α levels are used to treat diseases.
The molecule for regulating the level of HIF-3α or the pharmaceutical composition containing the molecule for regulating the level of HIF-3α according to claim 11, wherein the molecule is a chemical small molecule or a biological macromolecule, for example, selected from the group consisting of siRNA, miRNA, and antisense nucleic acid. , PROTAC drugs.
The molecule for regulating HIF-3α levels or a pharmaceutical composition containing a molecule for regulating HIF-3α levels according to claim 11 or 21, wherein the molecule is an agonist of HIF-3α;

Preferably, the molecule is OEA (oleoylethanolamine):
The molecule for regulating the level of HIF-3α or the pharmaceutical composition containing the molecule for regulating the level of HIF-3α according to claim 11 or 12, wherein the molecule is an antagonist or inhibitor of HIF-3α.
The molecule for regulating the level of HIF-3α or the pharmaceutical composition containing the molecule for regulating the level of HIF-3α according to any one of claims 11 to 14, wherein the disease is selected from the group consisting of infection and tumor.
The molecule for regulating HIF-3α levels or a pharmaceutical composition containing a molecule for regulating HIF-3α levels according to claim 15, wherein the infection is a viral infection, such as an infection caused by a pathogenic virus;

Preferably, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Herpesviridae;

Preferably, the Paramyxoviridae virus is measles virus;

Preferably, the Orthomyxoviridae virus is influenza A virus (such as H1N1);

Preferably, the Flaviviridae virus is hepatitis C virus;

Preferably, the Herpesviridae virus is a herpes virus or Epstein-Barr virus.
The molecule for regulating HIF-3α levels or a pharmaceutical composition containing a molecule for regulating HIF-3α levels according to claim 15, wherein the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, Liver cancer, kidney cancer, skin cancer, melanoma, glioma and sarcoma.
An anti-infection or anti-tumor method, comprising administering to a subject in need thereof an effective amount of a molecule that modulates HIF-3α levels, a pharmaceutical composition containing a molecule that modulates HIF-3α levels, or claims 8 or 9 The steps of the pharmaceutical composition.
The method of claim 18, wherein the molecule is a chemical small molecule or a biological macromolecule, for example, selected from the group consisting of siRNA, miRNA, antisense nucleic acid, and PROTAC drugs.
The method of claim 18 or 19, wherein the molecule is an agonist of HIF-3α;

Preferably, the molecule is OEA (oleoylethanolamine):
The method of any one of claims 18-20, wherein the molecule is an antagonist or inhibitor of HIF-3α.
The method of any one of claims 18-21, wherein active ingredient (a) and active ingredient in the pharmaceutical composition Ingredient (b) is administered to the subject simultaneously, sequentially or sequentially.
The method of any one of claims 18-22, wherein the disease is selected from the group consisting of infections and tumors.
The method of claim 23, wherein the infection is a viral infection, such as an infection caused by a pathogenic virus;

Preferably, the pathogenic virus is selected from the following families: Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Herpesviridae;

Preferably, the Paramyxoviridae virus is measles virus;

Preferably, the Orthomyxoviridae virus is influenza A virus (such as H1N1);

Preferably, the Flaviviridae virus is hepatitis C virus;

Preferably, the Herpesviridae virus is a herpes virus or Epstein-Barr virus.
The method of claim 23, wherein the tumor is selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, melanoma, glioma and sarcoma.
An anti-infection or anti-tumor drug screening method, which includes the following steps:

(1) Provide test molecules, HIF-3α (especially HIF-3αPAS-B) and HIF-3α overexpressing cells;

(2) Determine the binding between the test molecule and HIF-3α. If the two are combined, proceed to the next step;

(3) Treat the cells with the test molecule, measure the transcription level of HIF-3α downstream genes (such as HSPA6) in the cells, and compare with the negative control group; when the expression of the HIF-3α downstream genes is higher than In the case of a negative control group, the test molecule is determined to be an anti-infection or anti-tumor drug candidate.