Method for treating mood disorder

Abstract

Inhibitors of the PI4KIII [alpha] / FAM126 / TTC7 complex and methods of preventing or treating mood disorders with the same are provided.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202080067722.2, application date September 27, 2020, and invention name “A method for treating mood disorders”. The original application is a national phase application with international application number PCT / CN2020 / 118044. This international application claims priority to the international patent application with application number PCT / CN2019 / 108548 and application date September 27, 2019, and the Chinese patent application with application number CN202011009274.9 and application date September 23, 2020. Technical Field

[0002] The present invention relates to a method for preventing or treating mood disorders, comprising administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof. Background Art

[0003] Mood disorders are mental disorders represented by anxiety, depression or mania, which have tended to increase in recent years and have become a major social problem. Anxiety and depression are the most common mental disorders and are complications of other neuropsychiatric diseases, stroke, tumors, etc. More than 70% of dementia patients have depression and / or anxiety symptoms (Selbaek, G., K. Engedal and S. Bergh, The prevalence and course of neuropsychiatric symptoms in nursinghome patients with dementia: a systematic review. J Am Med Dir Assoc, 2013. 14 (3): p. 161-9).

[0004] At present, the overall efficacy of anti-anxiety and depression drugs in clinical practice is not ideal, especially for patients with severe anxiety and depression, neurodegenerative diseases and post-stroke depression, and anxiety and depression in patients with malignant tumors. The therapeutic effect is poor or basically ineffective, and they also have the disadvantages of slow onset of effect and widespread and serious side effects.

[0005] Prior to the present application, it was generally believed that decreased levels of phosphoinositide (PI) and the enzymatic activity of phosphoinositide-3 kinase (PI3K) were associated with depression. PI3K deficiency increases anxiety levels in mice, while several classes of traditional antidepressants increase the resynthesis of phosphoinositide in brain areas associated with anxiety and depression (see, for example: Di Paolo, G. and P. De Camilli, Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006. 443(7112): p. 651-7; Saito, T., et al., Polymorphism screening of PIK4CA: possible candidate gene for chromosome 22q11-linked psychiatric disorders. Am J Med Genet B Neuropsychiatr Genet, 2003. 116B(1): p. 77-83; Balla, A. and T. Balla, Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol, 2006. 16(7): p. 351-61).

[0006] Surprisingly, the inventors of the present application found that in various disease model mice, reducing the expression of PI4KIIIα or Efr3a used to anchor the PI4KIIIα / FAM126 / TTC7 complex by genetic methods, or applying PI4KIIIα inhibitors can reduce the anxiety or depression of mice. Therefore, the present application provides a potential target for anti-mood disorders (i.e., PI4KIIIα protein complex) and a series of potential drugs for treating mood disorders. Summary of the invention

[0007] In one aspect, the present application provides a method for preventing or treating mood disorders, comprising administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof.

[0008] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a small molecule compound, an antibody, an RNAi molecule or an antisense nucleic acid.

[0009] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an EFR3-specific inhibitor.

[0010] In some embodiments, the EFR3-specific inhibitor is an EFR3a-specific inhibitor or an EFR3b-specific inhibitor.

[0011] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a PI4KIIIα-specific inhibitor.

[0012] In some embodiments, the PI4KIIIα-specific inhibitor is a small molecule compound.

[0013] In some embodiments, the small molecule compound has a structure shown in formula (I) or a pharmaceutically acceptable salt thereof,

[0014]

[0015] wherein R1 is independently selected from (a) H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Alkylene -NH2, C 1-6 Alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH or heterocyclic, and optionally substituted by R2 or R3, wherein R2 and R3 are each independently selected from amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aromatic)amino, N,N-(C 1-6 Alkyl)2amino, C 3-6 Cycloalkyl, 6-12 membered aromatic or 3-12 membered heterocyclic, and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6cycloalkyl or Bn-O-substituted, and R4 is C 1-6 and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, R5 is selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy or C 1-6 haloalkyl, or

[0016] (b) R1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted,

[0017] Here, n is an integer from 0 to 5.

[0018] In some embodiments, each of the R1 groups is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, -As(O), N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino or -C(O)OR6, wherein n is an integer from 0 to 2, and R6 is C 1-6 of alkyl.

[0019] In some embodiments, each of the R1 groups is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or -As(O), wherein n is an integer from 0 to 2.

[0020] In some embodiments, each of the R1 is independently selected from H, deuterium, halogen, amino, C 1-6 Alkoxy or C1-6 A haloalkyl group wherein n is 1.

[0021] In some embodiments, the R1 is located at the ortho and / or para position to the -As(O) group.

[0022] In some embodiments, n is 0.

[0023] In some embodiments, the small molecule compound is selected from the group consisting of:

[0024]

[0025]

[0026]

[0027] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0028] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0029] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA).

[0030] In some embodiments, the RNAi molecule is 18-100 bases in length.

[0031] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0032] In some embodiments, the subject is a human or a mammal.

[0033] In some embodiments, the mood disorder is elevated mood, depressed mood, or cycling between elevated and depressed mood.

[0034] In some embodiments, the mood disorder is anxiety, depression, schizophrenia, or mania.

[0035] In some embodiments, the mood disorder is a mood disorder caused by a neurodegenerative disease, stroke, or malignancy.

[0036] In some embodiments, the mood disorder is anxiety or depression caused by a neurodegenerative disease.

[0037] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is administered orally, subcutaneously, intramuscularly, or intravenously.

[0038] In some embodiments, it further comprises diagnosing the subject with a mood disorder before administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to the subject.

[0039] In some embodiments, it further comprises administering a second agent to a subject in need thereof.

[0040] In some embodiments, the second agent is an agent used to treat a mood disorder.

[0041] In some embodiments, the second agent is an agent used to treat a neurodegenerative disease, stroke, or malignancy.

[0042] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is administered before, after, or simultaneously with the second agent.

[0043] In another aspect, the present application provides use of the PI4KIIIα / FAM126 / TTC7 complex inhibitor described herein in the preparation of a medicament for preventing or treating mood disorders.

[0044] In another aspect, the present application provides use of the PI4KIIIα / FAM126 / TTC7 complex inhibitor described herein for preventing or treating mood disorders, comprising administering an effective amount of the PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The figure shows the changes in body weight of rats in each group during the CUMS modeling and drug administration stages.

[0046] Figure 2 The results showed that the mRNA expression level of PI4KIIIα was positively correlated with the degree of depression.

[0047] Figure 3 The results show that the PI4KIIIα inhibitor PAO can prevent or alleviate the anxiety behavior of APP / PS1 mice (WT refers to wild-type mice, "APP / PS1 0" refers to APP / PS1 mice not treated with PAO, and "APP / PS1 0.3" refers to mice treated with PAO at a dose of 0.3 mg / kg body weight. "*" represents P<0.05)

[0048] Figure 4The results show the effects of PAO and Aricept on APP / PS1 mice in the water maze test.

[0049] Figure 5 Shown are the effects of PAO and Aricept on APP / PS1 mice in a novelty suppressed feeding experiment.

[0050] Figure 6 The figure shows the effect of reducing the mRNA expression of Efr3a on APP / PS1 mice in the black-white box experiment.

[0051] Figure 7 The effects of PAO derivatives PI04, PI05 and PI06 on ICR mice in the forced swimming test are shown. The black and gray bars are the sum of the immobility time within 4 minutes after the first and second swimming tests of each group of mice, respectively.

[0052] Figure 8 The results show that PAO has a preventive or alleviating effect on depression caused by neuronal damage in mice.

[0053] Fig. 9 The results show that PAO and its derivatives have an inhibitory effect on anxiety-like behavior in wild-type mice.

[0054] Fig.10 Shown is the interaction of Hyccin (FAM126), RBO, TTC7 and P4KIIIα in Drosophila. Fig.10 A: Coomassie blue staining of the immunoprecipitation of rbo-egfp transgenic and wild-type (wt) adult fruit fly head homogenates with anti-GFP antibody. The expression of RBO-GFP is driven by the rbo gene driver; Fig.10 B: Co-immunoprecipitation-immunoblotting (WB) showed that endogenous Hyccin, RBO and PI4KIIIα (PI4K) in adult wild-type Drosophila co-immunoprecipitated with each other; Fig.10 C: Western blot (WB) shows wild type and hyccin - / - ,rbo - / - ,ttc7 ＊ / ＊ Expression levels of RBO and Hyccin in the cell membrane and cytoplasm of mutant Drosophila embryos. RBO is only expressed on the cell membrane and is not present in the embryos of the three homozygous mutants mentioned above. Hyccin is mainly present in the cytoplasm. - / - and ttc7 ＊ / ＊ There was no significant change in the cytoplasmic levels of the mutants, indicating that the expression of Hyccin is independent of the expression of RBO and TTC7; Fig.10 D: Western blot (WB) showed that - / + and hyccin - / +The expression level of Hyccin in adult heterozygous mutant flies further showed that the expression of Hyccin was independent of RBO; Fig.10 E: WB shows wild type (wt), rbo - / + and hyccin - / + Heterozygous mutant adult flies, as well as control (ctrl) and heterozygous flies for four pi4kIIIα nonsense mutations (pi4k FQ88 / + ,pi4k FY24 / + ,pi4k GJ86 / + and pi4k GS27 / + ) Total levels of PI4KIIIα and RBO in adult flies. Four pi4kIIIα nonsense mutants were obtained by treating control flies (ctrl) with gene mutagen and screening. DETAILED DESCRIPTION

[0055] The following description of the present invention is intended only to illustrate various embodiments of the present invention. The specific embodiments described should not be construed as limiting the scope of the present invention. Without departing from the spirit and substance of the present invention, those skilled in the art may perform various equivalent substitutions, changes or variations, and it should be understood that these equivalent embodiments are also included herein. All documents cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0056] In one aspect of the present application, a method for preventing or treating mood disorders is provided, comprising administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof.

[0057] PI4KIIIα / FAM126 / TTC7 complex inhibitors

[0058] The term "PI4KIIIα / FAM126 / TTC7 complex" used in this application refers to a complex composed of three proteins, PI4KIIIα, FAM126 and TTC7, which is localized on the cytoplasmic membrane and participates in lipid phosphorylation in vivo. EFR3a or EFR3b is an important molecule that anchors the PI4KIIIα / FAM126 / TTC7 complex to the cytoplasmic membrane.

[0059] The term "PI4KIIIα / FAM126 / TTC7 complex inhibitor" used in this application refers to any substance that can reduce, decrease, or eliminate the activity of the PI4KIIIα / FAM126 / TTC7 complex. In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an inhibitor that inhibits the transcription or translation of the PI4KIIIα or FAM126 or TTC7 or EFR3 gene. In other embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an inhibitor that inhibits the ability of PI4KIIIα to phosphorylate lipids. In other embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an inhibitor that inhibits the formation of the PI4KIIIα / FAM126 / TTC7 complex. In other embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an inhibitor that inhibits the anchoring of the PI4KIIIα / FAM126 / TTC7 complex to the plasma membrane.

[0060] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is capable of reducing the activity of the PI4KIIIα / FAM126 / TTC7 complex by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

[0061] In this application, when "activity" is used with increase or decrease, it refers to the detected functional activity, which can be manifested as a change in content, or a change in functional activity while the content remains unchanged. In some embodiments, the activity is related to lipid phosphorylation. In some embodiments, the activity is related to mood disorders.

[0062] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a small molecule compound, an antibody, an RNAi molecule or an antisense nucleic acid.

[0063] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a small molecule compound.

[0064] The term "small molecule compound" as used herein refers to an organic compound with a molecular weight of less than 3000, 2500, 2000, 1500, 1000 or 500 Daltons, which may be natural or chemically synthesized.

[0065] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an antibody.

[0066] The term "antibody" used in this application includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody or antibody that can bind to a specific antigen. The term "antibody" in this application is intended to broadly cover conventional four-chain antibodies and less conventional antibodies that do not have four chains (e.g., antibodies that naturally lack light chains).

[0067] A conventional complete antibody is a heterotetramer, which contains two heavy (H) chains and two light (L) chains. The heavy chains of mammals can be divided into α, δ, ε, γ and μ, each of which consists of a variable region (V H ) and the first, second, and third constant regions (respectively C H1 , C H2 , C H3 );Mammalian light chains can be divided into λ or κ, each light chain consists of a variable region (V L) and a constant region. Conventional antibodies are "Y" shaped, and the neck of the "Y" structure is composed of the second and third constant regions of two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure contains the variable region and the first constant region of one of the heavy chains, which are combined with the variable region and constant region of a light chain. The variable regions of the light chain and heavy chain determine the binding of antigens. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light chain include LCDR1, LCDR2, LCDR3, and the CDRs of the heavy chain include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application can be named or identified by Kabat, IMGT, Chothia or Al-Lazikani nomenclature (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3): 651-63 (1985); Chothia, C and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252): 877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, 1989). Health, Bethesda, Maryland (1991); Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al, Immunome Research, 1 (3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). Among them, the three CDRs are separated by flanking continuous parts called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not related to antigen binding, but have a variety of effector functions.

[0068] Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. Depending on whether they contain α, δ, ε, γ and μ heavy chains, antibodies can be divided into five major categories or isoforms: IgA, IgD, IgE, IgG and IgM. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0069] In some embodiments, the antibody is a full-length antibody or an antigen-binding fragment.

[0070] The term "antigen binding fragment" as used in this application refers to an antibody fragment formed by an antibody fragment containing one or more CDRs but not having a complete antibody structure. Examples of antigen binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single domain antibodies (camelized single domain antibody) and nano antibodies. Antigen binding fragments can bind to the same antigen as the parent antibody.

[0071] The "Fab" fragment of an antibody refers to that portion of the antibody which is composed of a light chain (including the variable region and the constant region) and the variable region and the first constant region of a heavy chain bound together by a disulfide bond.

[0072] "Fab'" fragment refers to the Fab fragment including part of the hinge region.

[0073] "F(ab')2" fragment refers to a dimer of Fab'.

[0074] The "Fv" fragment of an antibody consists of the variable region of one light chain and the variable region of one heavy chain.

[0075] "Single-chain antibody molecule" or "scFv" refers to an engineered antibody in which the light chain variable region is directly connected to the heavy chain variable region or connected through a peptide chain. For detailed description, see, for example, Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988).

[0076] "ScFv dimer" refers to an aggregate formed by two scFvs.

[0077] "Camelized single domain antibody" (also called "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)") refers to an antibody containing two heavy chain variable regions but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74 (4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Although the light chain is missing, the camelized antibody has all the functions of antigen binding (see Hamers-Casterman C. et al., Nature. 363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. 54(1):39-47 (2002); Nguyen VK. et al., Immunology. 109(1):93-101 (2003)), the entire contents of which are incorporated into this application by reference.

[0078] "Nanobodies" are composed of a heavy chain variable region from a heavy chain antibody and two constant regions CH2 and CH3.

[0079] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0080] In some embodiments, the antibody is a murine antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0081] The term "fully human" as used in this application, when applied to an antibody or antigen-binding fragment, means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source such as a transgenic non-human animal utilizing a human antibody library, or other sequences encoding human antibodies.

[0082] The term "humanization" used in this application, when used for antibodies or antigen-binding fragments, refers to antibodies or antigen-binding fragments including CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. Because humanized antibodies or antigen-binding fragments have lower immunogenicity, they can be used as therapeutic agents for people in certain embodiments. In certain embodiments, the non-human animal is a mammal (e.g., mouse, rat, rabbit, goat, sheep, guinea pig, or hamster). In certain embodiments, the humanized antibody or antigen-binding fragment, except that the CDR sequence is non-human, is substantially all composed of human sequences.

[0083] The term "chimeric" as used in this application, when used for antibodies or antigen-binding fragments, refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species, while the remainder of the heavy chain and / or the light chain is derived from a different species. In some embodiments, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse or a rabbit).

[0084] In some embodiments, the antibodies described herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0085] In some embodiments, the antibodies described herein can be further labeled.

[0086] In some embodiments, the antibody described herein is a specific antibody for PI4KIIIα or FAM126 or TTC7 or EFR3. In some embodiments, the antibody described herein is a specific antibody for PI4KIIIα or EFR3.

[0087] In some embodiments, the antibodies described herein can inhibit the formation of the PI4KIIIα / FAM126 / TTC7 complex, inhibit the anchoring of the PI4KIIIα / FAM126 / TTC7 complex to the plasma membrane, or inhibit the phosphorylation activity of the PI4KIIIα / FAM126 / TTC7 complex.

[0088] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an RNAi molecule.

[0089] The term "RNAi molecule" as used herein refers to RNA or its analogs, which have sufficient sequence complementarity with a target RNA to guide RNA interference. In some embodiments, DNA that can be used to generate RNA is also included. RNA interference (RNAi) refers to a sequence-specific or selective process by which a target molecule (e.g., a target gene, protein, or RNA) is downregulated.

[0090] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA).

[0091] The term "small interfering RNA (siRNA)" used in this application refers to an RNA molecule, preferably a double-stranded molecule, having a length of about 10 to 50 nucleotides, preferably a length of about 15 to 25 nucleotides, more preferably a length of about 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, the chain optionally having a protruding end including, for example, 1, 2 or 3 protruding nucleotides (or nucleotide analogs), which can guide or mediate the degradation of RNA. Naturally occurring siRNAs are produced by longer (e.g., >25 nucleotides) dsRNA molecules by the RNAi machinery of the cell (e.g., Dicer or its homologues), and degrade mRNA by forming a RISC complex.

[0092] The term "short hairpin RNA (shRNA)" used in this application refers to an RNA molecule with a stem-loop structure, which includes a first region and a second region of complementary sequence, the degree of complementarity and the orientation of the regions are sufficient to allow base pairs to appear between the regions, the first region and the second region are connected by the loop region, and the loop is generated by the lack of base pairing between nucleotides (or nucleotide analogs) in the loop region.

[0093] The term "microRNA (microRNA or miRNA)" used in this application is a short, naturally occurring non-coding single-stranded RNA molecule with a length of about 16-26 nucleotides (nt) (e.g., about 16-29nt, 19-22nt, 20-25nt, or 21-23nt), which is usually involved in regulating gene expression in vivo. In eukaryotic cells, miRNA genes are transcribed into "primary products" (pri-miRNA) by DNA transcriptase II, and pri-miRNA is quickly processed into miRNA "precursors" (pre-miRNA) by a ribonuclease III (Drosha). Pre-miRNA is transported from the nucleus to the cytoplasm and then recognized and sheared into mature miRNA by another ribonuclease III (Dicer). Mature miRNA molecules are complementary to one or more mRNA parts and regulate protein expression. The sequences of known miRNAs can be obtained through public databases, such as the miRBase database ( www.mirbase.org), which provides information including miRNA sequence information, functional annotations, and predicted gene targets. In the present invention, miRNA also includes RNA molecules expressed in cells by artificially synthesized plasmids with structures and functions similar to natural miRNAs, which can target corresponding mRNAs like natural miRNA molecules and hinder their translation into proteins.

[0094] In some embodiments, the RNAi molecule is capable of reducing the expression of PI4KIIIα, for example, knocking down the PI4KA gene.

[0095] In some embodiments, the RNAi molecule has SEQ ID NO: 1 [5'-TGCTCATTAGCAGTAAAGA-3'].

[0096] In some embodiments, the RNAi molecule is 18-100 bases in length.

[0097] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0098] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an antisense nucleic acid.

[0099] The term "antisense nucleic acid" used in the present application includes nucleotides that are completely complementary to the target sequence and those nucleotides with one or more nucleotide mismatches, as long as the antisense nucleic acid can specifically hybridize with the target sequence. For example, the antisense nucleic acid of the present application includes polynucleotides with at least 70% or higher, preferably 80% or higher, more preferably 90% or higher, and even more preferably 95% or higher homology in a length of at least 15 consecutive nucleotides. Due to the formation of hybrids, target gene transcription and / or target mRNA translation are reduced or blocked. Standard methods related to antisense technology are described (see, for example, Melani et al., Cancer Res. (1991) 51: 2897-2901).

[0100] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an EFR3-specific inhibitor.

[0101] The term "EFR3 specific inhibitor" used in this application refers to any inhibitor that can specifically reduce, reduce, or eliminate the transcription or translation of the EFR3 (i.e., formyl-CoA reductase from yeast) gene, and / or the activity of the EFR3 protein. In some embodiments, the EFR3 specific inhibitor can reduce the activity of EFR3 by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more. In some embodiments, the activity of EFR3 refers to the activity of anchoring the PI4KIIIα / FAM126 / TTC7 complex to the cytoplasmic membrane.

[0102] In some embodiments, the EFR3-specific inhibitor is able to preferentially recognize the EFR3 protein in a complex mixture, and the binding constant of the inhibitor to the EFR3 protein is at least 2 times greater than the binding constant of the inhibitor to other non-specific binding proteins. In certain embodiments, the equilibrium dissociation constant of the EFR3-specific inhibitor to the EFR3 protein is less than or equal to 10 -5 or 10 - 6 In certain embodiments, the equilibrium dissociation constant of the EFR3-specific inhibitor with the EFR3 protein is less than or equal to 10 -6 or 10 -7 In certain embodiments, the equilibrium dissociation constant of the EFR3-specific inhibitor with the EFR3 protein is less than or equal to 10 -7 M or 10 -8 M. In some embodiments, the EFR3-specific inhibitor is an EFR3a-specific inhibitor or an EFR3b-specific inhibitor.

[0103] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a PI4KIIIα-specific inhibitor.

[0104] The term "PI4KIIIα specific inhibitor" used in this application refers to various substances that can specifically reduce, reduce, or eliminate the transcription or translation of the PI4KIIIα gene, and / or the activity of the PI4KIIIα protein. In some embodiments, the PI4KIIIα specific inhibitor can reduce the activity of PI4KIIIα by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more. In some embodiments, the activity of PI4KIIIα refers to the activity of PI4KIIIα phosphorylating lipids (e.g., phosphoinositides (PI)) (e.g., converting to 4-phosphoinositide phosphatidylinositol (PI4P)).

[0105] In some embodiments, the PI4KIIIα specific inhibitor is able to preferentially recognize the PI4KIIIα protein in a complex mixture, and the binding constant of the inhibitor to the PI4KIIIα protein is at least 2 times greater than the binding constant of the inhibitor to other non-specific binding proteins. In certain embodiments, the equilibrium dissociation constant of the PI4KIIIα specific inhibitor to the PI4KIIIα protein is less than or equal to 10 -5 or 10 -6 In some embodiments, the equilibrium dissociation constant of the PI4KIIIα specific inhibitor with the PI4KIIIα protein is less than or equal to 10 -6 or 10 -7 In some embodiments, the equilibrium dissociation constant of the PI4KIIIα specific inhibitor with the PI4KIIIα protein is less than or equal to 10 -7 M or 10 -8 M.

[0106] In some embodiments, the PI4KIIIα specific inhibitor inhibits PI4KIIIα at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 10,000-fold more strongly than PI4KII (e.g., PI4KIIα or PI4KIIβ). In some embodiments, the PI4KIIIα specific inhibitor inhibits PI4KIIIα at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 10,000-fold more strongly than PI4KIIIβ. In some embodiments, the PI4KIIIα specific inhibitor does not substantially inhibit PI4KII (e.g., PI4KIIα or PI4KIIβ), for example, its IC 50 Greater than or equal to 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM or 500 μM.

[0107] In some embodiments, the IC of a PI4KIIIα-specific inhibitor to PI4KIIIα is 50 Less than or equal to 100 μM, 80 μM, 50 μM, 30 μM, 20 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 0.5 μM, 0.2 μM, 0.1 μM, 0.05 μM, 0.02 μM, 0.01 μM, 0.005 μM, 0.002 μM or 0.001 μM.

[0108] In some embodiments, the PI4KIIIα-specific inhibitor is a small molecule compound.

[0109] In some embodiments, the small molecule compound described in the present application has a structure shown in formula (I) or a pharmaceutically acceptable salt thereof,

[0110]

[0111] wherein R1 is independently selected from (a) H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Alkylene -NH2, C 1-6 Alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH or heterocyclic, and optionally substituted by R2 or R3, wherein R2 and R3 are each independently selected from amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aromatic)amino, N,N-(C 1-6 Alkyl)2amino, C 3-6 Cycloalkyl, 6-12 membered aromatic or 3-12 membered heterocyclic, and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 cycloalkyl or Bn-O-substituted, and R4 is C 1-6 and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, R5 is selected from H, C 1-6 Alkyl, C2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy or C 1-6 haloalkyl, or

[0112] (b) R1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted,

[0113] In some embodiments, n is 0, 1, 2 or 3. In some embodiments, n is 0, 1 or 2. In some embodiments, n is 0 or 1.

[0114] In some embodiments, each of the R1 groups is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, -As(O), N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino or -C(O)OR6, wherein n is an integer from 0 to 2, and R6 is C 1-6 of alkyl.

[0115] In some embodiments, each of the R1 groups is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or -As(O), wherein n is an integer from 0 to 2.

[0116] In some embodiments, each of the R1 is independently selected from H, deuterium, halogen, amino, C 1-6 Alkoxy or C 1-6 A haloalkyl group wherein n is 1.

[0117] In some embodiments, the R1 is located at the ortho or para position to the -As(O) group. In some embodiments, the R1 is H.

[0118] As used herein, the term "substituted", when referring to a chemical group, means that one or more hydrogen atoms of the chemical group are removed and replaced with a substituent.

[0119] The term "substituent" as used herein has its ordinary meaning as known in the art and refers to a chemical moiety which is covalently attached or, where appropriate, fused to a parent group.

[0120] The term "C n -C m " represents a range of carbon atoms, wherein n and m are integers and the range of carbon atoms includes the endpoints (i.e., n and m) and each integer point therebetween. For example, C1-C6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms and 6 carbon atoms.

[0121] The term "alkyl" as used herein, whether as part of another term or used alone, refers to a saturated hydrocarbon group, which may be straight-chain or branched. n -C m "Alkyl" refers to an alkyl group having n to m carbon atoms. In certain embodiments, the alkyl group contains 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.

[0122] The term "alkenyl" used in the present application, whether as a part of other terms or used alone, refers to an unsaturated alkyl group, which can be straight or branched, with at least one carbon-carbon double bond. In certain embodiments, the alkenyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms. In certain embodiments, the alkenyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 carbon-carbon double bond. The example of alkenyl groups includes but is not limited to chemical groups such as vinyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl etc.

[0123] The term "alkynyl" used in the present application, whether as a part of other terms or used alone, refers to an unsaturated alkynyl group, which can be straight or branched, with at least one carbon-carbon triple bond. In certain embodiments, the alkynyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms. In certain embodiments, the alkynyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 carbon-carbon triple bond. The example of the alkynyl group includes but is not limited to chemical groups such as ethynyl, propynyl, butynyl etc.

[0124] The term "cycloalkyl" used in this application refers to a ring system alkyl consisting of at least 3 atoms. The term "nm-membered cycloalkyl" refers to a cycloalkyl having n to m members forming a ring. In addition, the ring may also have one or more double bonds, but does not have a completely conjugated system. In certain embodiments, the cycloalkyl has 3 to 8, 3 to 6, or 4 to 6 carbon atoms forming a ring. The example of cycloalkyl includes, but is not limited to, cyclopropane, cyclobutane, cyclopentyl, etc.

[0125] The term "heterocyclic radical" used in this application refers to a cyclic radical in which at least one atom in the ring system is a heteroatom and the remaining ring atoms are carbon atoms. The term "nm-membered heterocyclic radical" refers to a heterocyclic radical having n to m members forming a ring. The term "heterocyclic radical" used in this application includes heteroaryl and heterocycloalkyl. In addition, the ring can also have one or more double bonds. In certain embodiments, the heterocyclic radical is a saturated heterocycloalkyl. The example of heteroatoms includes, but is not limited to, oxygen, sulfur, nitrogen, phosphorus, etc.

[0126] The term "heterocycloalkyl" used in this application refers to a cycloalkyl group in which at least one atom in the ring system is a heteroatom and the remaining ring atoms are carbon atoms. The term "nm-membered heterocycloalkyl" refers to a heterocycloalkyl group having n to m members forming a ring. In addition, the ring can also have one or more double bonds, but does not have a completely conjugated system. In certain embodiments, heterocycloalkyl is a saturated heterocycloalkyl group. The example of heteroatoms includes but is not limited to oxygen, sulfur, nitrogen, phosphorus, etc. In certain embodiments, heterocycloalkyl has 3 to 8, 3 to 6 or 4 to 6 carbon atoms forming a ring. The example of heterocycloalkyl includes but is not limited to azetidine, aziridine, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholine, homopiperazine, etc.

[0127] The term "aryl" or "aromatic group" as used herein, whether as part of another term or used alone, refers to a mono- or poly-carbocyclic ring system radical having alternating double and single bonds between the carbon atoms forming the ring. n -C m "Aryl" refers to an aromatic group having n to m carbon atoms forming the ring. In certain embodiments, an aryl ring system has 6 to 12, 6 to 10, or 6 to 8 carbon atoms in one or more rings. In certain embodiments, an aryl ring system has 2 or more rings fused together. Examples of aryl groups include, but are not limited to, chemical groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.

[0128] The term "heteroaryl" used in this application refers to an aryl group in which at least one ring atom in an aromatic ring is a heteroatom and the remaining ring atoms are carbon atoms. The term "nm yuan heteroaryl" refers to a heteroaryl with n to m members forming a ring. The example of heteroatoms includes but is not limited to oxygen, sulfur, nitrogen, phosphorus, etc. In certain embodiments, heteroaryl can have 5 to 10, 5 to 8 or 5 to 6 members forming a ring. In certain embodiments, heteroaryl is a 5-yuan or 6-yuan heteroaryl. The example of heteroaryl includes but is not limited to furyl, thienyl, pyridyl, quinolyl, pyrrolyl, N-low alkyl pyrrolyl, pyridyl-N-oxide, pyrimidinyl, pyrazinyl, imidazolyl, indolyl, etc.

[0129] The term "alkoxy" as used herein, whether as part of another term or used alone, refers to a group of the formula "-O-alkyl". n -C m "Alkoxy" refers to an alkoxy group in which the alkyl portion has n to m carbon atoms. In certain embodiments, the alkyl portion has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxy groups include, but are not limited to, chemical groups such as methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

[0130] The term "haloalkyl" as used herein, whether as part of another term or used alone, refers to a group of the formula "-alkyl-X" where X is a halogen selected from fluorine, chlorine, bromine and iodine atoms. n -C m "Haloalkyl" refers to an alkyl group whose alkyl portion has n to m carbon atoms. In certain embodiments, the alkyl portion has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of haloalkyl groups include, but are not limited to, chemical groups such as halomethyl, haloethyl, halopropyl (e.g., n-halopropyl and isohalopropyl), t-halobutyl, and the like.

[0131] As used herein, the term "n-membered", where n is an integer, is generally used with a ring system to describe the number of atoms in the ring system that form the ring. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridinyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered aryl.

[0132] The term "halogen" as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0133] The term "cyano" as used herein refers to a group represented by the formula "-CN".

[0134] The term "hydroxyl" as used herein refers to a group represented by the formula "-OH".

[0135] The term "nitro" as used herein refers to a group represented by the formula "-NO2".

[0136] The term "amino" as used herein refers to a group represented by the formula "-NH2".

[0137] The term "carbamoyl" used herein refers to a group represented by the formula "-HNCONH2".

[0138] As used herein, the term "compound" is intended to include all stereoisomers (eg, enantiomers and diastereomers), geometric isomers, tautomers, and isotopes of the depicted structure.

[0139] The compounds described herein can be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended to be included. The compounds of the present application containing asymmetrically substituted carbon atoms can be separated in optically activated or racemic forms. How to prepare optically activated forms from optically non-active starting materials is well known in the art, such as by splitting racemic mixtures or by stereoselective synthesis. Multiple geometric isomers such as olefins, carbon-carbon double bonds can also be present in the compounds described herein, and all of these stable isomers have been considered in the present application. The present application describes the cis and trans geometric isomers of compounds and they can be separated in a mixture of isomers or in a single isomeric form.

[0140] In certain embodiments, the compounds described herein have the (R) configuration. In certain embodiments, the compounds described herein have the (S) configuration.

[0141] The racemic mixture of compound can be split by any of the multiple methods known in the art. Exemplary method includes fractional crystallization using chiral resolution acid, which is a kind of optically active salified organic acid. Suitable resolution reagent for fractional recrystallization method is, for example, optically active acid (such as, tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid D and L form) or various optically active camphorsulfonic acids. Other resolution reagents suitable for fractional crystallization method include N-methylbenzylamine, 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1, the stereoisomerically pure form of 2-diaminocyclohexane etc.

[0142] Resolution of the racemic mixture can also be performed by elution on a column packed with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0143] Compounds of the present application also include tautomeric forms. Tautomeric forms are caused by the exchange of single bonds and adjacent double bonds accompanied by the migration of protons. Tautomeric forms include tautomers of protons with isomeric protonation states of the same chemical formula and total charge. The example of proton tautomers includes keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs and cyclic forms, wherein protons can occupy two or more positions of heterocyclic systems, such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles and 1H- and 2H-pyrazoles. Tautomeric forms can be balanced or spatially locked into a form by suitable substitution.

[0144] The compounds of the present application may also include all isotopes of atoms present in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, deuterium and tritium.

[0145] In certain embodiments, the small molecule compounds of the present application can be obtained by organic synthesis. Any known organic synthesis technique can be used to prepare and the compounds of the present application can be synthesized according to a variety of possible synthetic pathways, including salts, esters, hydrates or solvates thereof.

[0146] The reaction of preparing the compounds of the present application can be carried out in a suitable solvent, and the technician in the field of organic synthesis can easily select the solvent. Suitable solvent can be substantially not reacted with the initial raw material (reactant), intermediate or product at the temperature at which the reaction is carried out (for example, it can be the temperature range from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in a solvent or a mixture of more than one solvent. According to the specific reaction steps, those skilled in the art can select a suitable solvent for the specific reaction steps.

[0147] The preparation of the compounds of the present application may involve the protection and deprotection of various chemical groups. Those skilled in the art can easily determine whether protection and deprotection are needed and select suitable protecting groups. Protecting group chemistry can be found in, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), the entire contents of which are incorporated herein by reference.

[0148] The reaction can be monitored by any suitable method known in the art. For example, spectroscopy, such as nuclear magnetic resonance spectroscopy (e.g. 1 H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or monitoring the formation of the product using chromatography, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). A variety of methods can be used by those skilled in the art to purify the compound, including high performance liquid chromatography (HPLC) (see, for example, "Preparative LC-MS Purification: Improved Compound Specific Method Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6), 874-883, the entire contents of which are incorporated herein by reference) and normal phase silica gel column chromatography.

[0149] In certain embodiments, the small molecule compounds of the present application can be purchased from commercial sources.

[0150] In some embodiments, the small molecule compound is:

[0151]

[0152]

[0153]

[0154]

[0155] The term "PAO" used in this application refers to a small molecule compound with an arsenic oxide group and a benzene ring as the basic structure, and its specific chemical structure is as follows:

[0156]

[0157] Mood disorders

[0158] The term "mood disorder" used in the present application refers to a state in which a person feels distressed due to abnormal emotions (feelings) that persist for a certain period of time and causes some kind of impairment in daily life.

[0159] In some embodiments, the mood disorder is elevated mood, depressed mood, or cycling between elevated and depressed mood.

[0160] The term "high spirits" used in this application refers to the main primary symptom of manic episodes. The typical manifestation is that the patient feels good about himself, so he appears to be elated, elated, and carefree. This high mood is very contagious and often makes people around him laugh. Some patients with manic episodes may be irritable as the main manifestation, and they will lose their temper at the slightest provocation, and may even engage in destructive and aggressive behavior.

[0161] The term "depression" as used in this application refers to a mental state that includes but is not limited to sadness and depression, which can cause destructive behavior. The patient feels depressed, distressed and sad, and the tone of the mood is low and gloomy. The depression of depressive episodes has two important characteristics: first, "significant and persistent", which means that this depressed mood is usually clearly felt by the person and (or) others, and the degree can range from being unhappy to heartbroken, and lasts for at least 2 weeks; second, this depression is "disproportionate to its actual situation", a distorted and exaggerated "depression", and it is difficult to explain with objective real-life circumstances.

[0162] The term "cycling between high and low moods" as used herein refers to the alternation of a mental state between high moods and low moods.

[0163] In some embodiments, the mood disorder is anxiety, depression, schizophrenia, or mania.

[0164] The term "anxiety disorder" used in this application includes panic disorder with or without agoraphobia, agoraphobia without a history of panic disorder, specific phobia, social phobia, obsessive-compulsive behavior disorder, post-traumatic stress disorder, acute stress disorder, generalized anxiety disorder, anxiety disorder due to general medical conditions, substance-induced anxiety disorders, and anxiety disorders not listed separately. As used herein, the term "anxiety disorder" includes those related disorders described in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (1994, American Psychiatric Association, Washington, DC). It should be recognized by those skilled in the art that there are alternative naming systems, disease classification systems, and classification systems for neurological and psychiatric disorders and, in particular, anxiety disorders, and these systems evolve as medical science develops. Therefore, the term "anxiety disorder" is intended to include similar disorders described in other diagnostic sources.

[0165] The term "depression" used in this application includes, for example, single-episode or recurrent major depression and dysthymic affective disorder, depressive neuropathy and functional depression. The depression of this application includes: melancholic depression, including anorexia, weight loss, insomnia and early awakening, and psychomotor retardation; atypical depression (or reactive depression), including increased appetite, excessive sleep, psychomotor restlessness or irritability, anxiety and phobia; seasonal affective disorder. As used herein, the term "depression" includes related disorders described in DSM-IV.

[0166] The term "schizophrenia" used in this application is a mental illness characterized by severe chronic mental impairment. Its symptoms appear in early adulthood, followed by a period of social impairment. Schizophrenia manifests itself as auditory and visual hallucinations, paranoia, delusions (positive symptoms), emotional retardation, depression, anhedonia, poverty of speech, memory and attention deficits, and social withdrawal (negative symptoms), etc.

[0167] The term "mania" as used in this application is a clinical condition characterized by elevated emotions or irritability, accompanied by high energy, increased speech, increased activity, and in severe cases, psychotic symptoms such as hallucinations, delusions, and tension symptoms. Manic episodes need to last for more than one week, generally presenting an episodic course, with an intermittent remission period of normal mental state after each episode, and most patients tend to have repeated episodes.

[0168] In some embodiments, the mood disorder is a mood disorder caused by a neurodegenerative disease, stroke, or malignancy.

[0169] In some embodiments, the mood disorder is anxiety or depression caused by a neurodegenerative disease.

[0170] Drug administration and medical use

[0171] The term "pharmaceutically acceptable" used in this application refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of humans and animals without excessive toxicity, stimulation, allergic reaction or other problems or complications within the scope of reasonable medical judgment, with a reasonable benefit / risk ratio. In certain embodiments, pharmaceutically acceptable compounds, materials, compositions and / or dosage forms refer to those approved by regulatory agencies (such as the U.S. Food and Drug Administration, the China Food and Drug Administration or the European Medicines Agency) or listed in generally recognized pharmacopoeias (such as the U.S. Pharmacopoeia, the Chinese Pharmacopoeia or the European Pharmacopoeia) for animals (more particularly for humans).

[0172] The term "object" used in this application can include humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Object" can also be livestock animals (for example, cattle, pigs, sheep, chickens, rabbits or horses), or rodents (for example, rats or mice), or primates (for example, gorillas or monkeys), or domestic animals (for example, dogs or cats). "Object" can be male or female, and can also be different ages. Human "object" can be Caucasian, African, Asian, Semitic, or other races, or hybrids of different races. Human "object" can be elderly, adult, teenager, child or infant.

[0173] In some embodiments, the subject described herein is a human or a mammal. In some embodiments, the subject described herein is a human or a non-human primate.

[0174] The PI4KIIIα / FAM126 / TTC7 complex inhibitor disclosed in this application can be administered by routes of administration known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection (including intravenous drip or intravenous infusion), intramuscular injection or intradermal injection) or non-injection administration (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration). In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor described in this application is administered orally, subcutaneously, intramuscularly or intravenously. In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor described in this application is administered orally.

[0175] The term "therapeutically effective amount" as used in this application refers to the amount of a drug that can alleviate or eliminate the disease or symptoms of the subject, or can preventively inhibit or prevent the occurrence of a disease or symptom. A therapeutically effective amount can be the amount of a drug that alleviates one or more diseases or symptoms of the subject to a certain extent; the amount of a drug that can partially or completely restore one or more physiological or biochemical parameters related to the cause of the disease or symptom to normal; and / or the amount of a drug that can reduce the likelihood of the occurrence of a disease or symptom. In some embodiments, the term "therapeutically effective amount" as used in this application refers to the amount of a drug that can alleviate or eliminate the mood disorder (e.g., anxiety or depression) of the subject.

[0176] The therapeutically effective dose of the PI4KIIIα / FAM126 / TTC7 complex inhibitor provided in this application depends on a variety of factors known in the art, such as body weight, age, past medical history, current treatment, health status of the subject and the intensity of drug interactions, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of disease development. A person skilled in the art (e.g., a doctor or veterinarian) may reduce or increase the dose accordingly according to these or other conditions or requirements.

[0177] In some embodiments, the method further comprises diagnosing the subject as having a mood disorder before administering an effective amount of the PI4KIIIα / FAM126 / TTC7 complex inhibitor to the subject. Whether a client has a mood disorder can be diagnosed using routine diagnostic methods and criteria used by psychiatric clinicians.

[0178] In some embodiments, the treating further comprises administering a second agent to a subject in need thereof.

[0179] In some embodiments, the second agent is an agent used to treat a mood disorder, including, but not limited to, a tricyclic antidepressant, a selective serotonin reuptake inhibitor (SSRI), or a serotonin / norepinephrine reuptake inhibitor (SNRI).

[0180] In some embodiments, the second agent is an agent used to treat a neurodegenerative disease, stroke, or malignancy. In some embodiments, agents treating neurodegenerative diseases include, but are not limited to, levodopa, carbidopa, selegiline, rasagiline, entacapone, tolcapone, amantadine, memantine, riluzole, bromocriptine, pergolide, apomorphine, ropinitrol, pramipexole, atropine, scopolamine, trihexyphenidyl, benzatropine, procyclidine, tacrine, donepezil hydrochloride, galanthamine hydrobromide, huperzine A, A) and Rivastigmine. In some embodiments, agents for treating stroke include but are not limited to dipyridamole, low molecular weight dextran, urokinase, heparin and aspirin. In some embodiments, agents for treating malignant tumors include but are not limited to cyclophosphamide, thiotepa, carmustine, cisplatin, carboplatin, methotrexate, pemetrexed, capecitabine, cytarabine, gemcitabine, paclitaxel, docetaxel and vinblastine.

[0181] In some embodiments, the PI4KIIIα / FAM126 / TTC7 complex inhibitor is administered before, after, or simultaneously with the second agent.

[0182] The present application also relates to the use of the PI4KIIIα / FAM126 / TTC7 complex inhibitor described in the present application in the preparation of a medicament for preventing or treating mood disorders, and the use of the PI4KIIIα / FAM126 / TTC7 complex inhibitor described in the present application for preventing or treating mood disorders.

[0183] Drug Screening

[0184] The present application also provides a method for screening PI4KIIIα / FAM126 / TTC7 complex inhibitors, comprising contacting an alternative drug with a protein or nucleic acid of PI4KIIIα, FAM126, TTC7 or EFR3a or a PI4KIIIα / FAM126 / TTC7 complex, and detecting whether the alternative drug can inhibit the formation or activity of the PI4KIIIα / FAM126 / TTC7 complex.

[0185] The present application also provides a method for screening drugs for preventing or treating mood disorders, comprising contacting an alternative drug with a protein or nucleic acid of PI4KIIIα, FAM126, TTC7 or EFR3a or a PI4KIIIα / FAM126 / TTC7 complex, and detecting whether the alternative drug can inhibit the formation or activity of the PI4KIIIα / FAM126 / TTC7 complex.

[0186] The present invention also includes the following embodiments:

[0187] Embodiment 1. A method for preventing or treating mood disorders, comprising administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof.

[0188] Embodiment 2. The method according to embodiment 1, wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a small molecule compound, an antibody, an RNAi molecule or an antisense nucleic acid.

[0189] Embodiment 3. The method according to embodiment 1, wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an EFR3-specific inhibitor.

[0190] Embodiment 4. The method according to embodiment 3, wherein the EFR3-specific inhibitor is an EFR3a-specific inhibitor or an EFR3b-specific inhibitor.

[0191] Embodiment 5. The method according to embodiment 1, wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a PI4KIIIα-specific inhibitor.

[0192] Embodiment 6. The method according to embodiment 5, wherein the PI4KIIIα-specific inhibitor is a small molecule compound.

[0193] Embodiment 7. The method according to Embodiment 2 or 6, wherein the small molecule compound has a structure shown in Formula (I) or a pharmaceutically acceptable salt thereof,

[0194]

[0195] wherein R1 is independently selected from (a) H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Alkylene -NH2, C 1-6 Alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH or heterocyclic, and optionally substituted by R2 or R3, wherein R2 and R3 are each independently selected from amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aromatic)amino, N,N-(C 1-6 Alkyl)2amino, C 3-6 Cycloalkyl, 6-12 membered aromatic or 3-12 membered heterocyclic, and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 cycloalkyl or Bn-O-substituted, and R4 is C 1-6 and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, R5 is selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy or C 1-6 haloalkyl, or

[0196] (b) R1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted,

[0197] Here, n is an integer from 0 to 5.

[0198] Embodiment 8. The method according to embodiment 7, wherein each of the R1 is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, -As(O), N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino or -C(O)OR6, wherein n is an integer from 0 to 2, and R6 is C 1-6 of alkyl.

[0199] Embodiment 9. The method according to embodiment 7, wherein each of the R1 is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or -As(O), wherein n is an integer from 0 to 2.

[0200] Embodiment 10. The method according to embodiment 7, wherein each of the R1 is independently selected from H, deuterium, halogen, amino, C 1-6 Alkoxy or C 1-6 A haloalkyl group wherein n is 1.

[0201] Embodiment 11. A method according to any one of Embodiments 7-10, wherein R1 is located at the ortho and / or para position to the -As(O) group.

[0202] Embodiment 12. The method according to embodiment 11, wherein n is 0.

[0203] Embodiment 13. The method according to embodiment 6, wherein the small molecule compound is selected from the group consisting of the following compounds:

[0204]

[0205]

[0206]

[0207]

[0208] Embodiment 14. The method according to embodiment 2, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0209] Embodiment 15. The method according to embodiment 2 or 14, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

[0210] Embodiment 16. The method according to embodiment 2, wherein the RNAi molecule is small interfering RNA (siRNA), short hairpin RNA (shRNA) or micro RNA (miRNA).

[0211] Embodiment 17. A method according to embodiment 2 or 16, wherein the RNAi molecule is 18-100 bases in length.

[0212] Embodiment 18. The method according to any one of embodiments 2 and 16-17, wherein the RNAi molecule is modified to enhance its stability.

[0213] Embodiment 19. The method of embodiment 1, wherein the subject is a human or a mammal.

[0214] Embodiment 20. The method of embodiment 1, wherein the mood disorder is elevated mood, depressed mood, or cycling between elevated and depressed mood.

[0215] Embodiment 21. The method of embodiment 1, wherein the mood disorder is anxiety, depression, schizophrenia or mania.

[0216] Embodiment 22. A method according to embodiment 1, wherein the mood disorder is a mood disorder caused by a neurodegenerative disease, stroke or malignant tumor.

[0217] Embodiment 23. A method according to embodiment 1, wherein the mood disorder is anxiety or depression caused by a neurodegenerative disease.

[0218] Embodiment 24. The method according to embodiment 1, wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is administered orally, subcutaneously, intramuscularly or intravenously.

[0219] Embodiment 25. The method according to embodiment 1, further comprising diagnosing the subject as having a mood disorder before administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to the subject.

[0220] Embodiment 26. The method of embodiment 1, further comprising administering a second agent to a subject in need thereof.

[0221] Embodiment 27. The method of embodiment 26, wherein the second agent is an agent used to treat a mood disorder.

[0222] Embodiment 28. The method of embodiment 26, wherein the second agent is an agent for treating a neurodegenerative disease, a stroke, or a malignancy.

[0223] Embodiment 29. A method according to embodiment 26, wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is administered before, after or simultaneously with the second agent.

[0224] Embodiment 30. Use of a PI4KIIIα / FAM126 / TTC7 complex inhibitor in the preparation of a medicament for preventing or treating mood disorders.

[0225] Embodiment 31. Use of a PI4KIIIα / FAM126 / TTC7 complex inhibitor for preventing or treating mood disorders, comprising administering an effective amount of the PI4KIIIα / FAM126 / TTC7 complex inhibitor to a subject in need thereof.

[0226] Example

[0227] Example 1: Antidepressant efficacy of oral administration of PAO in the rat model of chronic mild unpredictable stress

[0228] 1.1 Test Principle

[0229] The chronic unpredictable mild stress (CUMS) model is a rat depression model proposed by Willner et al. in 1987, inspired by the chronic unpredictable stress described by Katz, and has been appropriately modified. It is a model widely used in the literature. This model more realistically simulates the "difficulties" encountered by people in their daily lives. In this model, the variability and unpredictability of stress factors are the key to the success of model making. The order of more than ten stress factors is randomly selected throughout the experiment, so that the animals cannot predict the occurrence of stimulation. This model is highly effective and basically meets the requirements of the depression model. It can be used to study problems that other models are unable to solve. The reduction in the amount of sucrose water consumed after long-term mild stress stimulation reflects the central symptom of endogenous depression, namely lack of pleasure, and also simulates the symptoms of other major depressive disorders, such as decreased motor ability and social interaction ability, decreased exploratory behavior ability, deficient aggressive ability, and decreased sexual behavior ability. Chronic stress-mediated behavioral abnormalities can persist for several months, and long-term use of antidepressants can correct these abnormal behaviors, while other types of psychiatric drugs are ineffective.

[0230] 1.2 Purpose of the experiment

[0231] The purpose of this study was to observe the effect of oral administration of PAO on the sugar water preference of rats in the chronic mild unpredictable stress model, so as to determine whether PAO has a good antidepressant effect.

[0232] 1.3 Test materials

[0233] 1.3.1 Test samples

[0234] Name: PAO

[0235] Preparation method: Weigh the required amount of PAO, prepare it into a stock solution with a concentration of 30 mg / ml using dimethyl sulfoxide (DMSO), and then dilute it with water.

[0236] Oral administration by gavage: The dosage range is 0.1-3.0 mg / kg, and the volume of the dosing solution is 0.5 ml per 100 g body weight.

[0237] 1.3.2 Positive Control

[0238] Name: Venlafaxine Hydrochloride Sustained Release Capsules (Enosi)

[0239] Specification: 150mg / capsule

[0240] Preparation method: Take 2 venlafaxine capsules (150 mg / capsule), crush them, suspend them in 0.5% CMC-Na after ultrasonication, and make the final volume to 100 ml with 0.5% CMC-Na.

[0241] 1.3.3 Experimental animals

[0242] Species: Wistar rat

[0243] Number and gender of animals used: 60, male

[0244] Body weight: 200-220g (body weight before grouping)

[0245] Source: Shanghai Slake Laboratory Animal Co., Ltd.

[0246] 1.4 Experimental methods

[0247] 1.4.1 Animal grouping

[0248] After acclimatization for 5 days before the test, healthy SD rats weighing 220-250 g were selected as test animals and divided into groups as shown in Table 1 below.

[0249] Table 1. Animal groups

[0250] Group Dosage (mg / kg) Number of animals Blank control (drinking water) 0 10 CUMS model group (drinking water) 0 10 Venlafaxine 15 10 PAO high dose oral administration 3.0 10 PAO medium dose 1.5 10 PAO oral administration low dose 0.75 10

[0251] 1.4.2 Animal modeling and drug administration methods

[0252] 1.4.2.1 Basic sugar water preference test

[0253] Rats were adaptively raised for 1 week, and then a basal sugar water preference test was performed. All animals were given 1% sucrose water within the first 24 hours: two bottles were filled with 1% sucrose water and placed in the left and right corners of the cage. In the next 24 hours, 1% sucrose water and ordinary drinking water were given at the same time: one bottle contained 1% sucrose water, and the other bottle contained ordinary drinking water, which were still placed in the left and right corners of the cage. Then they were fasted and deprived of water for 23 hours, and then a sugar water preference test was performed: 1% sucrose water and ordinary drinking water were given at the same time, and the amount of sucrose water and ordinary drinking water consumed by the animals within 1 hour was measured by weighing the drinking bottles. The animals were evenly grouped as shown in Table 1 according to their body weight, fluid consumption, and sugar water consumption, and then subjected to chronic mild unpredictable stress stimulation ("CUMS").

[0254] 1.4.2.2 CUMS depression model

[0255] CUMS depression model was established in all groups except the blank control group. More than ten stress factors were given to rats alternately every week, so that the rats could not foresee the duration of each stress factor stimulation and the stimulation that would be given next. The stimulation factors of the experiment were as follows: 2 times of 7-hour flash stimulation; 1 time of 17-hour unclean bedding environment (150ml of water was poured on the bedding); 1 time of 3-hour and 1 time of 5-hour intermittent noise stimulation; 1 time of night light (light lasted for 36 hours); 1 time of 7-hour and 1 time of 22-hour mouse cage tilting; 1 time of 17-hour group housing (5 mice in a cage), 1 time of 17-hour unfamiliar odor environment housing; 1 time of 7-hour unfamiliar foreign environment; 3 times of fasting lasting 19, 22 and 24 hours respectively, and 2 hours of limited feed supply after each fasting; 4 times of water deprivation lasting 17, 19, 20 and 21 hours respectively, and an empty bottle was placed for 1 hour after each water deprivation (Table 2).

[0256] Table 2. CUMS stimulation schedule

[0257]

[0258] 1.4.2.3 Method of administration

[0259] In the first two weeks of CUMS stimulation modeling, rats were not given PAO. PAO or venlafaxine was orally administered to rats on the first Monday after two weeks of CUMS modeling, and rats in the blank group and model group were also given drinking water. The doses were given according to Table 1, once a day, and continued until the end of the CUMS procedure. CUMS stimulation continued during the drug administration.

[0260] 1.4.3 Depressive behavior indicators - sugar water preference test

[0261] The fluid consumption was measured after fasting and water deprivation every week (measured on Friday), and the body weight (recorded on Thursday), total fluid intake, water intake, and sugar intake of each animal were recorded. The percentage of sugar preference for each mouse (calculated as follows: [preference = (sugar intake / total fluid intake) × 100%]) and sugar consumption per gram of body weight were calculated. The experimental period was 9 weeks.

[0262] 1.4.4 Statistical methods

[0263] SPSS software was used for statistical analysis, and the data are presented as mean ± standard error. Indicates that the body weight differences between the other groups of animals and the blank group were statistically tested by two-way ANOVA; the differences in sugar preference values ​​between the other groups and the model group after 1 and 2 weeks of administration, as well as the differences in sugar preference values ​​between the other groups and the blank control group were tested for significance using Unpaired t test, P<0.05 is marked with *, and P<0.01 is marked with **.

[0264] 1.5 Test results

[0265] 1.5.1 CUMS modeling and changes in body weight after drug administration

[0266] When initially grouped, the weights of the animals in each group were similar, with no significant differences. As CUMS progressed, except for the untreated blank group, the weight gain of the animals in other groups slowed down significantly because they were all subjected to CUMS depression modeling (Table 3, Figure 1 Since the first week of stimulation, the body weights of the animals in the other groups stimulated by CUMS were lower than those in the blank control group in weekly measurements (Table 3, Figure 1 ), indicating that the use of CUMS for depression modeling in this experiment is very effective and well simulates the weight loss during the onset of depression.

[0267] Table 3. Body weight of rats in each group during CUMS modeling and drug administration

[0268]

[0269] 1.5.2 Antidepressant efficacy of multiple doses of PAO

[0270] At the initial grouping, the animals in each group had similar sugar preference. After 6 weeks of administration, 3 and 0.75 mg / kg doses of PAO and venlafaxine both showed a trend of increasing the sugar preference values ​​of rats, which was significantly different from the model group rats (P<0.05 or P<0.01), and their sugar preference values ​​were similar to those of the blank control group rats (P>0.05) (Table 4). The results of this experiment showed that oral administration of PAO can have a significant antidepressant effect in the rat CUMS depression model, and its onset time is slightly later than that of venlafaxine administered by oral gavage.

[0271] Table 4. Effects of PAO on sugar water preference in CUMS rats (n=10 per group, )

[0272]

[0273] 1.6 Conclusion

[0274] Oral administration of PAO at 3 and 0.75 mg / kg or the positive drug venlafaxine once a day for 6 weeks showed an increase in the sugar preference of CUMS depression model rats and increased the sugar preference value to a level similar to that of normal rats, reflecting the antidepressant effect. Oral administration of PAO at a dose of 1.5 mg / kg also tended to increase the sugar preference.

[0275] Example 2: Further verification and mechanism study of the anti-anxiety and anti-depressant efficacy of PAO

[0276] 2.1 Mouse strains, breeding, and reproduction

[0277] The mouse strains used in the present invention include C57 / 6B and ICR wild-type mice, Efr3a - / + Mouse (the second exon of Efr3a is unconditionally knocked out, resulting in subsequent genetic code shift), Pi4kaGt (RRO073) Byg / + (Pi4k - / + ) mice and APP / PS1 mice.

[0278] Pi4kaGt(RRO073) Byg / + The mouse was Pi4kaGt (RRO073) from the Mutant Mouse Resource and Research Center, USA. Byg / + ] mice (MMRRC, Cat.#016351-UCD) and C57 / 6B mice are backcrossed for more than 5 generations. A transposon is inserted into the PI4KA (encoding PI4KIIIα) gene of the mice and polyA is introduced, which can terminate the transcription of PI4KA prematurely. APP / PS1 mice are double transgenic mice with the genotype of B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax(MMRRC ID 034832-JAX), which is a model mouse for Alzheimer's disease (AD).

[0279] In this application, all kinds of mice were raised in standard-sized cages, with 5 mice per cage, and occasionally 4 or 6 mice per cage. Unless otherwise specified, mice were free to eat and drink water.

[0280] 2.2 Establishment of hormone-induced anxiety and depression model in mice

[0281] ICR mice were orally administered with corticosterone (GC, i.e. 11β,21-dihydroxypregnene-3,20-dione), and a mouse depression model was obtained one month later. Hydroxypropyl-β-cyclodextrin (β-CD) was used as a solubilizer to prepare an aqueous solution with a GC concentration of 35μg / ml, in which the β-CD concentration was 0.45%.

[0282] 60 male C57B / 6 WT mice were divided into six groups, with 10 mice in each group. When the mice were 3 months old, the drinking water of four groups of mice was replaced by the above GC solution, and the other two groups continued to be given ordinary drinking water, and the weight changes and water intake of the mice were recorded weekly. After one month, two groups were randomly selected from the mice fed with GC solution and water, respectively, and given PAO solution by gavage. PAO was administered by gavage at regular intervals every day, at a dose of 0.2 and 0.4 mg / kg body weight, with a total volume of 300 μl. One month after administration, when the mice were 5 months old, behavioral experimental analysis of the mice was performed.

[0283] 2.3 Configuration of PAO or its derivatives

[0284] Preparation of PAO mother solution: Take PAO ≤ 30 mg and put it into 1.0 ml of pure DMSO solvent, sonicate or oscillate until PAO is completely dissolved. Then dilute with water to the required concentration, and the final concentration of DMSO is 0.1%.

[0285] In certain embodiments, PAO or its derivative is dissolved in medium chain triglycerides, the stock solution concentration is 1.0 mg / g, and when administered intragastrically, it is diluted with medium chain triglycerides to a specific concentration.

[0286] 2.4 Mouse behavioral experimental methods

[0287] 2.4.1 Forced Swimming Test (FST)

[0288] Forced swimming test (by placing the experimental animals in a confined environment (such as water), the animals struggle desperately in this environment to try to escape but cannot escape, thus providing an unavoidable oppressive environment. After a period of time, the animals show a typical "immobility state", a depression model called "behavioral despair state". The experiment is carried out in a transparent plastic cylinder with an inner diameter of 15 cm and a height of 30 cm. The water is filled to a height of 15 cm. The height can be adjusted appropriately, so that the mouse's tail cannot touch the bottom and the mouse cannot jump out of the water tank. During the experiment, the water temperature should be kept between 22℃-25℃. The forced swimming image acquisition system should be placed in front of the FST device, at a certain distance from the water tank and can clearly record the movement of the mouse's limbs in the water. Each time is required to be timed for 6 minutes, and the immobility time of the mouse from the 2nd minute to the 6th minute is analyzed. After the experiment, the mouse is taken out and dried in time. If the mouse drowns during the experiment, it should be taken out in time and the FST experiment on the mouse should be stopped. During the experiment, an absolutely soundless environment should be created and maintained. The longer the mouse remains immobile for a certain period of time, the deeper the degree of depression of the mouse.

[0289] 2.4.2 The light / dark box test (LDB)

[0290] The black-and-white box experiment is an experiment that uses the animal's dark-loving characteristics (dark-moving box) and exploratory characteristics (white-moving box) to evaluate animal anxiety behavior. LDB consists of a nine-square white box (27×27cm) and a six-square black box (18×27cm). The whole (45×27×27cm) is a rectangular box without a lid. The white box and the black box are separated by a board with a small window (7.5×7.5cm). An image acquisition system is placed directly above the box to record the distance and trajectory of the mouse's shuttle in the black-and-white box within a set time, the number of times it enters and exits the black-and-white box, and the time it stays in the black box or white box respectively. The more frequently the mouse enters and exits the black-and-white box, the longer the shuttle distance in each box, or the longer the time it stays in the black box, the deeper the anxiety of the mouse. Before the experiment, the mouse cage should be wrapped with tin foil, but there must be an opening for air exchange. The experiment should be started after half an hour of light blocking, in order to allow the mice to adapt to the dark environment in advance and eliminate the experimental panic and anxiety of the mice. Then, the mice are placed one by one in the center of the white box or black box, facing the small window, and the number of times the mice shuttle between the black and white boxes within 5 minutes and 30 seconds, the shuttle distance in the black and white boxes, and the stay time in each box are recorded. After the image is collected, behavioral analysis is performed. After each experiment with a mouse, 70% ethanol is used to thoroughly wipe the position in the black and white box that the mouse can touch. After the alcohol smell is completely dissipated, the next mouse experiment is started to avoid the smell of the previous mouse causing unnecessary misleading and interference to the behavior of the next mouse. During the entire experiment, an absolutely soundless environment should be created and maintained.

[0291] 2.4.3 The elevated plus maze (EPM)

[0292] The elevated plus maze is one of the experimental methods for evaluating the anxiety response of rodents. Compared with the detection of anxiety behavior of mice caused by noxious stimulation (such as electrical stimulation, noise stimulation, dietary deprivation and exposure to predator odor, etc.), this experiment has the advantages of simple operation and low harm, and can intuitively reflect the conditioned response of mice. EPM consists of two open arms and two closed arms, which are crossed in a cross shape. The crossing part is the central area, about 75 cm from the ground. The principle is that when facing new things (open arms), mice will be curious to explore, and they have a dark nature (closed arms), and there will be a conflict between exploration and avoidance between the two, resulting in anxiety. During the experiment, a single mouse was placed in the central area facing the closed arm, and the number of times the mouse entered and exited the open arm and the closed arm within 5 minutes and 30 seconds, as well as the time it stayed in each arm, was recorded. The more frequently the mouse entered the open arm and the closed arm or the longer it stayed in the closed arm, the deeper the anxiety of the mouse. After each experiment, 70% ethanol should be used to thoroughly wipe the places in the elevated plus maze that the mouse can touch. The next experiment should be started after the smell of alcohol has dissipated to avoid unnecessary misleading and interference of the smell of the previous mouse on the behavior of the next mouse. During the entire experiment, an absolutely silent environment should be created and maintained.

[0293] 2.4.4 Novelty-suppressed feeding test (NSF)

[0294] The novelty suppression feeding experiment is to detect the conflict between feeding and fear of the new environment in rodents in a hungry state after fasting. The experimental venue is a 30×30cm square, 20cm high white opaque plexiglass box, with a normal mouse food placed in the center of the bottom of the box. After the mice have fasted for 24 hours, a single mouse is randomly placed in a corner of the glass box with its head facing the corner of the glass box. Observe the activities of the mouse in the glass box until the mouse starts to gnaw the food, take out the mouse, and record the time from the mouse being placed in the glass box to the mouse starting to gnaw the food (feeding latency). Unless otherwise designed or specified, if the mouse does not gnaw the food for 5 minutes, the mouse is also taken out, and the feeding time of the mouse is recorded as 5 minutes. The longer the feeding latency, the deeper the anxiety level.

[0295] 2.4.5 Chronic unpredictable moderate stress (CUMS) model

[0296] The experiment lasted for 8-10 weeks, and two stress factors were randomly selected from a variety of stress stimuli and given to mice every day. The stress stimuli included: clamping the tail for 15 minutes, strong light stimulation for 4 hours, noise for 10 minutes, tilting the cage at 45° for 24 hours, hanging upside down for 10 minutes, limiting the activity space for 4 hours, unclean bedding environment for 24 hours (pour 150ml of water into the bedding), night light (light lasts for 36 hours), unfamiliar foreign environment for 7 hours; fasting for 24 hours (remove the bedding during fasting); continuous water deprivation for 24 hours (remove the bedding during water deprivation). However, continuous water deprivation was only once a week, and water deprivation and unclean bedding environment did not overlap. After continuous water deprivation, each mouse was weighed, and then the preference for sugar water was measured. When measuring the preference for sugar water, a single mouse was placed in a new cage with two bottles, one containing pure water and the other containing 1% sugar water. The weight of the two bottles was weighed in advance. After the mouse drank water for 15 minutes, the positions of the two bottles were swapped, and the mouse was allowed to drink water for another 45 minutes. Take out the two water bottles and weigh the weight of the two bottles. After the experiment, the mice were immediately given sufficient water and food.

[0297] 2.4.6 Morris water maze

[0298] The Morris Water Maze (MWM) device consists of a water maze, a water maze image acquisition system, and a software analysis system. The water maze is mainly composed of a water pool (diameter = 1.2m) and a round transparent platform (diameter = 5cm) with adjustable height and movable position. The water in the pool contains titanium dioxide. Before each experiment, it should be stirred and mixed so that the mice cannot visually find the platform in the pool. The water temperature is maintained at around 20°C during the experiment. The behavior of the mice in the pool is collected by a camera placed above the water maze and transmitted to a computer connected to it for online (on-line) or offline (off-line) analysis and storage.

[0299] The water maze experiment mainly consists of three stages: adaptation training, acquisition training, and exploration training, and sometimes there is also a reverse acquisition training stage. The brief steps are as follows:

[0300] Adaptation training: One day before the start of acquisition learning, adjust the water temperature to an appropriate temperature, mix the water pool, and place a platform 0.5 cm above the water surface in the third quadrant of the pool. Put the mouse into the pool facing the pool wall and let it swim freely for 1 minute. Take the mouse out and wipe it dry. If the mouse can climb the platform within 1 minute, let the mouse stay on the platform for 0.5 minutes.

[0301] Acquisition training: generally lasts for 7 days, but can be longer or shorter. Divide the pool into 4 quadrants, place the platform in the middle of quadrant one, and randomly place the mice in the water from the four quadrants facing the pool wall. Paste four different markers, triangle, square, four-pointed star and circle, on the four walls of the pool to facilitate the memory and learning of the mice. Record the time it takes for the mice to find the underwater platform. If the time reaches 60 seconds, guide the mice to the platform, let them stay on the platform for 30 seconds, then take them out and wipe them dry; if the platform is found before 60 seconds, record the time, and let them stay for 30 seconds after finding the platform before taking them out and wiping them dry. If the mice have the ability to learn and remember, the time it takes to find the platform will become shorter and shorter every day.

[0302] Exploration training: On the second day after the last acquisition training, remove the platform in quadrant one and start a 60-second exploration training. Place the mice in the water from one end of quadrants three and four (farthest from quadrant one), and record the percentage of the total time spent by the mice in quadrant one (the quadrant where the platform was previously placed) as a test indicator of spatial memory. After the experiment is terminated, the mice should be taken out and dried in time. Generally, the mice that can find the platform in the least time in each quadrant during acquisition training or the mice that spend a greater percentage of time in quadrant one during exploration training have stronger learning and memory abilities. During the experiment, an absolutely silent environment should be created and maintained.

[0303] Reverse acquisition training: After the exploration phase, the platform was placed in the quadrant opposite to the original position. Each animal was trained in 4 quadrants per day for a total of 4 times, with each training lasting 60 seconds. After the animal found the hidden platform, it was allowed to stay on the platform for 30 seconds. If it did not find the platform, it was guided to step onto the platform and stay for 30 seconds. The interval between animal training was 15-20 minutes.

[0304] 2.5 Quantitative PCR analysis of PIK4IIIα mRNA levels in mouse brain

[0305] 2.5.1 Main instruments, equipment and reagents

[0306] Table 5. Main instruments, equipment and reagents

[0307]

[0308] 2.5.2 Primer information

[0309] Table 6. Primer information

[0310]

[0311]

[0312] 2.5.3 Experimental procedures

[0313] RAN extraction: add 1000μl RNAiso Plus to each brain tissue sample (half brain), grind with a tissue lyser (10 rpm × 10 sec / cycle × 10 cycles), let stand at room temperature for 5 min, and centrifuge at 12000g for 10 min at 4℃; take 900μl of the supernatant and add it to a fresh 1.5ml EP tube, add 200μl of chloroform to each, mix well, and let stand at room temperature for 5 min; centrifuge at 12000g for 15 min at 4℃, transfer 400μl of the supernatant to a new 1.5ml EP tube, add 800μl of isopropanol to each, let stand at room temperature for 10 min; centrifuge at 12000g for 10 min at 4℃, and discard the supernatant; add 1ml / tube of 75% ethanol, mix gently, centrifuge at 7500g for 5 min at 4℃, and discard the supernatant; let the precipitate dry at room temperature, and add 50μl of H2O to each tube to dissolve.

[0314] Reverse transcription: Use a DNA-free reverse transcription kit, 20 μl system. Take 1 μg RNA in a 200 μl PCR tube, add 5×gDNA eraser buffer 2 μl / tube, gDNA eraser 1 μl / tube, and H2O to 10 μl. Digest DNA at 42℃ for 2 minutes. Add PrimeScript RT Enzyme MIX I 1 μl / tube to the reaction solution.

[0315] tube, RT primer mix 1μl / tube, 5×primeScript buffer2 (for real time) 4μl / tube, RNase free H2O 4μl. Perform reverse transcription at 37℃ 15min, 85℃ 5sec, 4℃. The obtained cDNA can be used immediately or stored at -20℃ for future use.

[0316] Real-time quantitative PCR: cDNA was diluted 10 times with RNase free H2O. The reaction system was TB Green PremixEx Taq II 12.5μl, bidirectional primers (10μM) 1μl each, cDNA template 2μl, RNase free H2O 8.5μl, and the total volume was 25μl. The reaction procedure used a two-step method: 95℃ pre-denaturation for 30sec; PCR reaction at 95℃ for 5sec, 60℃ for 30sec, for a total of 40 cycles; and finally, the melting curve program was run. Three technical replicates were performed for each sample.

[0317] 2.5.4 Data analysis: The △CT method was used to perform relative quantification of target genes.

[0318] 2.6 Data Analysis and Statistics

[0319] SPSS software was used for comparative analysis of the data. The statistical data in the article are presented as mean ± standard error. The standard for significant difference in data is p value < 0.05.

[0320] 2.7 Experimental Results

[0321] 2.7.1 PI4KIIIα mRNA level in mouse brain is positively correlated with depression severity

[0322] Thirty-five male 2-month-old ICR mice were subjected to the first forced swimming test to experience unavoidable oppression. On the seventh day, these mice underwent a second forced swimming test, and the time each mouse spent in the "immobile state" for the last 4 minutes of the total 6 minutes of the second forced swimming was recorded. Then, the brain of each mouse was quickly removed, the total RNA in the brain tissue was extracted, and reverse transcription was performed to obtain cDNA. Quantitative PCR was performed on the cDNA of PI4KIIIα and the internal reference beta-actin of each mouse, and the △CT method was used for relative quantification to analyze and calculate the amount of PI4KIIIα RNA relative to the internal reference RNA in the brain tissue of each mouse. After eliminating the data with an "immobile state" time of less than 10s or greater than 160s, a dot matrix was made with the remaining "immobile state" time of each mouse and the relative amount of PI4KIIIα RNA as the X and Y axis values, respectively ( Figure 2 ). The results showed that the mRNA level of PI4KIIIα in the mouse brain was linearly positively correlated with the severity of depression. This experimental result suggests that PI4KIIIα plays a role in regulating or mediating the severity of depression.

[0323] 2.7.2 PI4KIIIα inhibitor alleviates anxiety and depression in APP / PS1 mice

[0324] Nine male APP / PS1 mice and nine wild-type male mice from the same litter were obtained. PAO solution (dose: 0.3 mg / kg body weight) was gavaged once a day from Monday to Friday for both groups of mice, and the drug was stopped on Saturday and Sunday. Nine male APP / PS1 mice of the same age were gavaged with a solution without PAO at the same time. Two months later, the three groups of mice underwent 8 days of water maze training and a memory test the next day for other experimental purposes. When the mice reached 10 months old, each mouse was transferred to an uncovered opaque plexiglass box with a length, width and height of 15 cm, and a modified household mosquito swatter was placed at the bottom of the box. When the mouse was in the center of the mosquito swatter, it was given a 1-second electric shock. Afterwards, it was returned to the home cage. After 7 days, an elevated plus maze experiment was performed, and the time each mouse spent in the open arm was recorded, and the percentage of the time spent in the open arm to the total time spent in the elevated plus maze was calculated. Figure 3The results showed that the percentage of time spent in the open arms by APP / PS1 mice not treated with PAO was significantly lower than that of wild-type mice, while the percentage of time spent in the open arms by APP / PS1 mice treated with PAO was not significantly different from that of wild-type mice. Figure 3 As shown, PAO can prevent or alleviate the anxiety symptoms in APP / PS1 mice.

[0325] In order to explore whether there is an inevitable interdependence between the therapeutic effects of drugs on APP / PS1 anxiety behavior and the therapeutic effects on learning and memory disorders of APP / PS1 mice, we tested the therapeutic effects of PAO and Aricept (Aricept, donepezil hydrochloride) on APP / PS1 anxiety and learning and memory disorders. Thirty-two 4-month-old male APP / PS1 mice of the same litter, the offspring of APP / PS1 and C57B / 6, were obtained and randomly divided into two groups. PAO (at a dose of 0.1 mg / kg body weight, this group of mice is called APP / PS1 PAO mice) and Aricept (at a dose of 1.0 mg / kg body weight, this group of mice is called APP / PS1 Aricept mice) were given by gavage every day from Monday to Saturday. In addition, 16 4-month-old male WT mice and 20 APP / PS1 mice of the same litter were also gavaged with 0.1% DMSO aqueous solution. After 5 weeks, a water maze test was performed. As Figure 4 As shown, untreated APP / PS1 mice performed the worst in the water maze, while WT mice performed the best. PAO-treated mice performed significantly better than untreated APP / PS1 mice in both acquisition training and reverse acquisition training, indicating that PAO significantly improved the learning and memory abilities of APP / PS1 mice. The performance of Aricept-treated mice in acquisition training was not significantly different from that of untreated APP / PS1 mice, but their performance in reverse acquisition training was the same as that of PAO-treated mice, and was also significantly better than that of untreated mice, indicating that Aricept significantly improved the learning and memory abilities of APP / PS1, but the therapeutic effect was not as good as that of PAO.

[0326] One week after the water maze test, mice in each group entered the novelty suppression feeding experiment. Figure 5As shown in the figure, the latency of untreated APP / PS1 mice to eat was significantly longer than that of WT mice, while the latency of PAO-treated APP / PS1 mice to eat was not significantly different from that of WT mice, indicating that PAO can significantly reduce the anxiety level of APP / PS1 mice; however, the anxiety level of Aricept-treated APP / PS1 mice was not significantly different from that of untreated APP / PS1 mice, and was even higher; indicating that although Aricept can significantly improve the learning and memory ability of APP / PS1 mice, it cannot reduce the anxiety level of APP / PS1 mice. Therefore, whether learning and memory can be improved is not necessarily related to whether anxiety can be improved.

[0327] 2.7.3 Downregulation of PI4KIIIα in wild-type mice can prevent or alleviate depression and anxiety symptoms

[0328] Get Pi4k - / + WT and Pi4k in the offspring of 2-month-old wild type (WT) and C57 / 6B - / + Mice (15 each) were transferred to the behavioral laboratory for 1 week of adaptation. The CUMS experiment began from the second week. The weight of WT mice began to stagnate and even decrease from the third week of CUMS; - / + Mice can continue to grow. From the 6th week of CUMS, Pi4k - / + There was a significant difference between the WT mice and the WT mice (Table 7). In terms of sugar water preference, before stress stimulation and in the first 2 weeks of CUMS, both groups of mice had a clear preference for sugar water, and there was no significant difference. From the third week of CUMS, the preference of both groups of mice for sugar water decreased week by week, but the WT group was more sensitive to sugar water than Pi4k. - / + The WT group showed a faster decline in sugar water preference than the Pi4k group at the 7th week of CUMS. - / + The experimental results show that down-regulating PI4KIIIα expression can prevent or alleviate depressive symptoms.

[0329] On the day after the CUMS experiment, mice were given sufficient water and food. They were fasted, given water, and their bedding was removed at 9 a.m. on the second day. A novelty-suppressed feeding experiment was performed on the third day. The results showed that the average feeding latency of the WT group was significantly lower than that of the Pi4k group. - / + The length of the group (WT: 212 ± 45 s, Pi4k - / + :154±27s, T-Test, P<0.05). The experimental results show that down-regulating PI4KIIIα expression can prevent or alleviate anxiety symptoms.

[0330] Table 7. WT and Pi4K - / + Body weight of mice during CUMS (n=15, mean±sem)

[0331]

[0332] "*" indicates significant difference compared with the corresponding WT mice, p < 0.05

[0333] Table 8. Effect of Pi4k deficiency on sugar water preference in CUMS mice (n=15, mean±sem)

[0334]

[0335] "*" and "**" indicate significant differences compared with the corresponding WT mice, p<0.05, p<0.01

[0336] 2.7.4 Downregulation of PI4KIIIα can prevent or alleviate depression and anxiety symptoms in neurodegenerative disease model mice.

[0337] To test whether downregulating PI4KIIIα expression can prevent or alleviate other diseases, especially anxiety and depression associated with neurological diseases, we obtained Pi4k - / + The offspring of APP / PS1 and APP / PS1;Pi4k at 4 months of age were mated with APP / PS1 in C57 / 6B genetic background. - / + "15 mice in each group were subjected to CUMS test, and the preference of the two groups of mice for sugar water was measured every week. As shown in Table 9, APP / PS1; Pi4k - / + The decrease in mice's preference for sugar water was significantly slower than that of APP / PS1 mice, indicating that downregulating PI4KIIIα expression can prevent or alleviate the depressive symptoms of APP / PS1 mice.

[0338] Table 9 Effect of downregulating PI4KIIIα on sugar water preference of APP / PS1 mice subjected to CUMS (n=15, mean±sem)

[0339]

[0340] "*" and "**" indicate significant differences compared with the corresponding APP / PS1 mice, p<0.05, p<0.01

[0341] Similarly, on the day after the CUMS experiment, mice were given sufficient water and food, and were fasted, given water, and their bedding was removed at 9 a.m. on the second day. A novelty-suppressed feeding experiment was performed on the third day. - / + The average latency of mice to eat was significantly shorter than that of the APP / PS1 group (APP / PS1: 242s, Pi4k - / +:183s, T-Test, P<0.05). The experimental results showed that down-regulating PI4KIIIα expression can prevent or alleviate the anxiety symptoms of APP / PS1 mice.

[0342] 2.7.5 Downregulation of Efr3a can prevent or alleviate depression and anxiety symptoms in neurodegenerative disease model mice.

[0343] Get APP / PS1 and Efr3a - / + The offspring of the mating littermates WT, Efr3a - / + , APP / PS1, APP / PS1; Efr3a - / + The number of mice was 15, 4, 14 and 9 respectively. When the mice reached 8 months old, the black and white box experiment was performed. Figure 6 The results showed that compared with wild-type mice (WT), the number of grid crossings (walking distance) of 8-month-old APP / PS1 mice in the white box increased significantly, while APP / PS1; Efr3 - / + There was no significant difference between the mice and WT mice, but it was significantly less than that of APP / PS1 mice. This suggests that the anxiety level of APP / PS1 mice is significantly higher than that of WT mice, and knocking out one copy of the Efr3a gene can significantly prevent or alleviate the anxiety level of APP / PS1 mice.

[0344] Example 3: Inhibitory effects of PAO and its derivatives on anxiety and depression in mice

[0345] This experiment tested the inhibitory effects of the PI4KIIIa inhibitor PAO and its derivatives on anxiety and depression in wild-type mice without obvious other disease symptoms or neural damage.

[0346] 3.1 PAO derivatives inhibit depressive behavior in wild-type mice

[0347] We also tested the effects of PAO derivatives on ICR mice in the forced swimming test. For example, the derivatives PI04, PI05 and PI06 were generated after the -H at the para position of the PAO benzene ring was replaced by -F, -CF3 and -I respectively. These three PAO derivatives were first prepared as a stock solution with a concentration of 1.0 mg / g using medium-chain triglycerides-MIGLYOL 812N (IOIOleo GmbH). The same oil was diluted in 3 steps to a solution of 0.033 mg / ml before oral administration to mice every day, and then administered by oral administration at a dose of 0.3 mg / kg body weight. The control group (veichle) was only given medium-chain triglycerides.

[0348] 80 2-month-old ICR mice were randomly divided into 4 groups. After acclimating to the forced swimming laboratory for 1 week, they were subjected to the first 6-minute forced swimming test. The total immobility time (immobile time) in the next 4 minutes was recorded. After the mice with a total immobility time of less than 10 seconds and greater than 160 seconds in each group were eliminated, 16, 14, 17 and 15 mice were left in the veichle (blank oil), PI04, PI05 and PI06 groups, respectively. Then, each group was gavaged once a day at a dose of 0.3 mg / kg body weight for 6 consecutive days. On the 7th day, a second 6-minute forced swimming test was conducted, and each mouse was given the same dose once 60 minutes before the forced swimming test. The immobility time of each mouse in the next 4 minutes was recorded. As Figure 7 As shown, there was no significant difference in the immobility time of mice given blank oil in the first and second forced swimming tests, while the immobility time of mice in the PI04, PI05 and PI06 groups in the second forced swimming test was significantly shorter than that in the first forced swimming test, indicating that PAO derivatives PI04, PI05 and PI06 can alleviate the behavioral manifestations of anxiety and depression.

[0349] 3.2PAO prevents or alleviates anxiety and depression caused by neuronal damage

[0350] The small molecule compound 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP) is dehydrogenated by monoamine oxidase B (MAO-B) to generate MPP + Ions inhibit mitochondrial enzyme complex I, causing cytotoxicity and even cell death. + Ions are specifically taken up by dopamine neurons in the substantia nigra, and MMP + Blocks complex I of the mitochondrial respiratory chain, leading to neurodegeneration in dopamine neurons.

[0351] Fifty 4-month-old C57B6 male mice were randomly divided into a blank group (Blank) of 10, a placebo group (PD-ctrl) after nerve injury of 20, and a PAO treatment group (PD-PAO) after nerve injury of 20. MPTP was first dissolved with DMSO and then diluted with normal saline, and probenecid was dissolved with a 5% sodium bicarbonate aqueous solution. First, each mouse in the placebo group and the treatment group was intraperitoneally injected with MPTP (supplier: CSN Pharm) once a day, 25 mg / kg, for 5 consecutive days, and then stopped for two days, and then injected again for 5 consecutive days according to the method of the first week. In the third week, each mouse was first intraperitoneally injected with probenecid (250 mg / kg) once a day, followed by an injection of MPTP (25 mg / kg) once a day for 5 consecutive days. The blank group was only injected with the corresponding solvent in the first three weeks. In the fourth week, the blank group and the placebo group were intragastrically administered with 0.1% DMSO solution every day, while the treatment group was intragastrically administered with PAO, 0.3 mg / kg (PAO dissolved in 0.1% DMSO aqueous solution), every day for 7 consecutive days.

[0352] On the 6th day of the fourth week, all animals were fasted but provided with water. On the 7th day, a sugar water preference test was conducted. Figure 8 A shows that the blank group mice have a clear preference for sugar water, the placebo group mice have a significantly reduced preference for sugar water (One way ANOVA, p < 0.05), and the treatment group still prefers sugar water, with no significant difference from the blank group. After the sugar water preference experiment, injection and gavage of mice in each group were stopped. On the 6th day of the sixth week, all animals were fasted, but water was provided; on the 7th day, the sugar water preference experiment was conducted again, and the results are as follows Figure 8 As shown in B, the three groups of animals all had a clear preference for sugar water, with no significant differences among them.

[0353] During the above experiment, a total of 12 mice died in the placebo group, 6 mice died in the treatment group, and no mice died in the blank group.

[0354] 3.3PAO and its derivatives inhibit anxiety-like behavior in wild-type mice

[0355] 42 3-month-old ICR male mice were randomly divided into 2 groups. The first group of 22 mice was a placebo group (vehicle), which was gavaged with a placebo (0.1% DMSO aqueous solution) every day. The second group was a PAO treatment group, which was gavaged with a 0.1% DMSO aqueous solution of PAO every day, with a dose of 0.3 mg / kg body weight, for 12 consecutive days. On the 12th day, a novelty suppression feeding test (NSF) was performed. 24 hours before the NSF test, all animals were prohibited from eating, but water was provided. The results are shown in Figure 2. Fig. 9As shown in A, after entering the new environment, the placebo group mice needed an average of 201.6±21.5 seconds (n=22) to start eating, while the PAO treatment group only needed an average of 159.6±20.3 seconds (n=20), which was significantly shorter than the placebo group (T Test, p<0.05). Therefore, PAO can inhibit the anxiety behavior of wild-type mice in a new environment.

[0356] Another 47 3-month-old ICR mice were randomly divided into 3 groups. The first group of 16 mice was the placebo group (vehicle), which was gavaged with a placebo (0.1% DMSO aqueous solution) every day. The second group of 15 mice and the third group of 16 mice were PI05 and PI06 treatment groups, respectively, which were gavaged with 0.1% DMSO aqueous solution of PI05 and PI06 every day, with a dose of 0.3 mg / kg body weight, for 7 consecutive days. On the 7th day, a novelty suppression feeding test (NSF) was performed. 24 hours before the NSF experiment, all animals were prohibited from eating, but water was provided. The results are shown in Figure 2. Fig. 9 As shown in B, after entering the new environment, the placebo group mice needed an average of 216.6±21.2 seconds (n=16) to start eating, while the PI05 and PI06 treatment groups needed an average of 194.6±21.7 seconds (n=15) and 154.2±21.2 seconds (n=16), respectively. The latency of eating in the PI06 treatment group was significantly shorter than that in the placebo group (one way ANOVA, p<0.05), and there was a trend of shortening in the PI05 treatment group.

[0357] Therefore, PAO and its derivatives can inhibit the anxiety behavior of wild-type mice in a new environment.

[0358] Example 4. Effect of Inhibition of PI4KIIIα, TTC7, Hyccin and RBO on Expression of PI4KIIIα Complex

[0359] Experimental Materials and Methods

[0360] Drosophila strains and genetics

[0361] Standard Drosophila medium was used to culture the emerging Drosophila adults in a 12 / 12 hr light / dark cycle at 25°C.

[0362] The fruit fly strains used are: rbo 2 / Cyo-GFP(rbo 2 is the rbo gene knockout mutation, Cyo-GFP is a balance chromosome expressing green fluorescent protein GFP, rbo - / + ), rbo 2 / rbo 2 ;rbo-egfp / rbo-egfp(rbo 2The mutants homozygous for the rbo-egfp transgene and the rbo-egfp transgene (the Drosophila strain is referred to as rbo-gfp) (the rbo-egfp transgene is a recombinant DNA constructed from a genomic DNA fragment containing the entire rbo, and a DNA sequence encoding green fluorescent protein (GFP) is inserted before the stop codon of rbo, so that the rbo-egfp transgene expresses an RBO-GFP fusion protein), P[lacW]l(2)k14710 k15603 / Cyo-GFP (ttc7 gene mutant formed by the insertion of transposon P[lacW], also known as ttc7 - / + ), 104577 / Cyo-GFP (104577 is a chromosome mutation formed by the deletion of the hyccin gene and its surrounding DNA fragments, also known as hyccin - / + ),pi4k GS27 / + ,pi4k FY24 / + 、pi4k FQ88 / + 、pi4k GJ86 / + (They are heterozygous for 4 nonsense mutations in the gene encoding PI4KIIIα protein, all of which have a balance chromosome FM7 expressing green fluorescent protein, Gr-GFP, all constructed by the Schupbach laboratory at Princeton University in the United States).

[0363] Western blotting and co-immunoprecipitation experiments

[0364] 500 (for immunoprecipitation) and 100 (for protein immunoblotting) fruit fly heads of each genotype were collected and processed uniformly. The fruit fly heads were placed on ice and lysed with pre-cooled Tris buffer containing 1% NP40 and protease inhibitors to prepare a homogenate. Co-Immunoprecipitation (Co-IP) kit (ThermoScientific) kit instructions were used. The experimental products of co-immunoprecipitation were separated using SDS-PAGE gel and stained with Coomassie Brilliant Blue. The primary antibodies used included: anti-GFP (Casico), anti-RBO and PI4KIIIα (Zhang et al., J Neurosci., 2017, pages 4928-4941), anti-Hyccin (produced by Abmart (Shanghai) using FAM126 fragment RLPPIKNPRQ as antigen), anti-neuroglin (DSHB, clone BP104), anti-β-actin and anti-Tubulin (Cell Signaling).

[0365] Isolation of PM fragments from tissue lysates

[0366] The Drosophila brain tissue was placed on ice and treated with pre-cooled Tris buffer containing 1% NP40 and protease inhibitors. The lysate was then collected and centrifuged at 15,000 g for 1 hour at 4°C. The supernatant was transferred to a new EP tube and the precipitate was resuspended using the same Tris lysis buffer as above. The supernatant and precipitate resuspended were subjected to SDS-PAGE gel and protein immunoblot analysis.

[0367] Statistical analysis

[0368] Data were analyzed using GraphPad Prism software. All data are expressed as mean ± SEM and were statistically significant when p < 0.05 (*p < 0.05, **p < 0.01 and ***p < 0.001).

[0369] result

[0370] PI4KIIIα, TTC7, Hyccin and RBO interact with each other on the cell membrane to form a protein complex - PI4KIIIα complex, and are all indispensable parts of the PI4KIIIα complex.

[0371] Coomassie blue staining of SDS-PAGE gel showed ( Fig.10 A): In the homogenate of fruit fly heads expressing RBO-GFP fusion protein (rbo-gfp), anti-GFP antibody can co-immunoprecipitate PI4KIIIα, TTC7, Hyccin and RBO-GFP proteins, but not in the homogenate of fruit fly heads that do not express RBO-GFP fusion protein. Protein profiling analysis can identify PI4KIIIα, TTC7 and Hyccin proteins from the proteins co-immunoprecipitated with RBO-GFP fusion protein (Table 10). Consistent with this, protein profiling analysis of anti-RBO antibodies can co-immunoprecipitate PI4KIIIα, TTC7, Hyccin and RBO proteins from wild-type fruit fly head homogenates (Table 11); and Western blotting of SDS-PAGE gels can confirm that PI4KIIIα, Hyccin and RBO proteins are co-immunoprecipitated with each other ( Fig.10 B).

[0372] Western blot of SDS-PAGE gel showed ( Fig.10C): RBO protein is only expressed on the cell membrane, while Hyccin protein is present in both the cell membrane and the cytoplasm, but is mainly located in the cytoplasm; knocking out the hyccin, rbo, and ttc7 genes separately can lead to RBO deficiency, and knocking out the rbo and ttc7 genes separately also leads to the deficiency or significant reduction of Hyccin protein in the cell membrane, but does not significantly change the expression of Hyccin protein in the cytoplasm. This indicates that the expression of PI4KIIIα, TTC7, Hyccin, and RBO proteins on the cell membrane is interdependent and is an indispensable part of the PI4KIIIα complex. However, the expression of Hyccin in the cytoplasm is independent of TTC7 and RBO.

[0373] Western blot of SDS-PAGE gel showed ( Fig.10 D): Knocking out one copy of hyccin can significantly reduce the total expression of Hyccin, but knocking out one copy of rbo has little effect on the total expression of Hyccin.

[0374] Western blot of SDS-PAGE gel showed ( Fig.10 E): Knocking out one copy of PI4KIIIα can also significantly reduce the expression of RBO, further demonstrating the interdependence of the components of the PI4KIIIα complex.

[0375] Table 10. Mass spectrometry analysis of specific RBO-GFP interacting proteins co-immunoprecipitated with GFP antibody in homogenates of RBO-GFP and WT flies

[0376] PepCount CoverPercent MW(kd) RBO-GFP 98 52.3％ 122 PI4KIII 186 35.5％ 244 TTC7 77 43.1％ 95 Hyccin 33 32.7％ 70

[0377] Table 11. Mass spectrometry analysis of RBO-interacting proteins co-immunoprecipitated with RBO antibodies (but not control proteins) in wild-type Drosophila homogenates

[0378] PepCount CoverPercent MW(kd) RBO 93 44.8％ 95 PI4KA 215 37.8％ 244 TTC7 55 30.6％ 95 Hyccin 20 25.4％ 70

Claims

1. A method for preventing or treating a mood disorder, comprising diagnosing the subject with a mood disorder, and then administering an effective amount of a PI4KIIIα / FAM126 / TTC7 complex inhibitor to the subject in need thereof, wherein the mood disorder is anxiety or depression. 2 . The method according to claim 1 , wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a small molecule compound, an antibody, an RNAi molecule or an antisense nucleic acid. 3 . The method according to claim 1 , wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is an EFR3-specific inhibitor. The method according to claim 3 , wherein the EFR3-specific inhibitor is an EFR3a-specific inhibitor or an EFR3b-specific inhibitor. The method according to claim 1 , wherein the PI4KIIIα / FAM126 / TTC7 complex inhibitor is a PI4KIIIα-specific inhibitor. The method according to claim 5 , wherein the PI4KIIIα-specific inhibitor is a small molecule compound.

7. The method according to claim 2 or 6, wherein the small molecule compound has a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, in, R1 is independently selected from (a) H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Alkylene -NH2, C 1-6 Alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH or heterocyclic, and optionally substituted by R2 or R3, wherein R2 and R3 are each independently selected from amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aromatic)amino, N,N-(C 1-6 Alkyl)2amino, C 3-6 Cycloalkyl, 6-12 membered aromatic or 3-12 membered heterocyclic, and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 cycloalkyl or Bn-O-substituted, and R4 is C 1-6 and optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, R5 is selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy or C 1-6 haloalkyl, or (b) R1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic, C 1-6 Alkyl, C 2-6 Alkynyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, Here, n is an integer from 0 to 5.

8. The method according to claim 7, wherein each of the R1 is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, -As(O), N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino or -C(O)OR6, wherein n is an integer from 0 to 2, and R6 is C 1-6 of alkyl.

9. The method according to claim 7, wherein each of the R1 is independently selected from H, deuterium, halogen, nitro, cyano, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl or -As(O), wherein n is an integer from 0 to 2.

10. The method according to claim 7, wherein each of the R1 is independently selected from H, deuterium, halogen, amino, C 1-6 Alkoxy or C 1-6 A haloalkyl group wherein n is 1.

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