A method of reducing fat in a fatty liver by perfusion ex vivo with a pi4kb inhibitor

Abstract

The present invention relates to a method of ex vivo reduction of steatotic liver fat, the method comprising perfusing a steatotic liver graft in a perfusion step with a perfusion liquid comprising a specified PI4KB inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Description

Technical Field

[0001] This invention relates to a method for reducing fatty liver fat in vitro by perfusion of PI4KB inhibitor.

[0002] This application claims the rights of European application 24166005.9, filed on 25 March 2024, the contents of which are incorporated herein by reference. Background Technology

[0003] The catabolism of major nutrients glucose, amino acids, and fatty acids (FA) is a central function in almost all cell types to maintain their various cellular activities. Of these nutrients, FA has the highest energy density and thus constitutes an important long-term cellular energy substrate. Due to the toxicity of FA at high concentrations, most of them are esterified into triglycerides (TG) and stored in lipid droplets (LD). To avoid nutrient shortages during starvation, TG reserves are mobilized to release FA, thereby meeting cellular energy needs through lipolysis or the autophagic degradation of LD (i.e., lipophage). Lipolysis occurs sequentially by fatty triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL), hydrolyzing TG into FA and glycerol, while lipophage is controlled by lysosomal acid lipase (LAL). Although LD can be produced in almost any cell type to store excess intracellular FA, adipose tissue is the organ with the highest levels of LD formation and storage. In the presence of a sufficient glucose supply, insulin signaling increases lipogenesis while inhibiting lipolysis to promote lipid storage in adipose tissue. Conversely, hormones (adrenaline) or sympathetic neurons (norepinephrine) stimulate adipocyte lipolysis to supply fatty acids to other organs during fasting.

[0004] Of the three lipases involved in lipolysis, HSL is activated via phosphorylation of protein kinase A (PKA) activated by catecholamine signaling. Conversely, insulin inhibits this process via cAMP degradation mediated by phosphodiesterase 3B (PDE3B). However, HSL loss results in a mild phenotype in both humans and mice. Therefore, liver-specific depletion of HSL does not affect hepatic triglyceride (TG) levels, and in adipose tissue, HSL loss leads to age-dependent hepatic steatosis due to progressive lipodystrophy. In contrast, ATGL plays a broader role, as evidenced by significant LD accumulation in various tissues from both humans and mice. Atgl Loss of function. Furthermore, liver-specific [effects] in mice. AtglKnockout of ATGL promotes hepatic LD accumulation, while adipose tissue-specific Atgl knockout shows a significant reduction in hepatic neutral lipid levels, highlighting the role of ATGL in providing intracellular and extracellular FA. ATGL function is regulated at multiple levels to meet the complex physiological needs of energy homeostasis. Transcriptionally, insulin signaling is inhibited by suppressing FOXO1. ATGL mRNA synthesis. At the post-translational level, catecholamine and glucagon signaling are transmitted through... PKA and CAMKII Phosphorylation of ATGL at Ser406 in adipocytes and hepatocytes promotes ATGL enzyme activity. Furthermore, ATGL abundance can be regulated by proteasome-mediated degradation following polyubiquitination, via various E3 ligases such as COP1 and PEX2.

[0005] The Golgi apparatus is defined by a tethered stack of pools containing numerous intracellular functions, including protein modification and folding, vesicle formation, cytoskeleton organization, and lipid metabolism. To perform these processes, the Golgi apparatus binds to transmembrane integrative proteins or recruits cytoplasmic proteins through interactions with Golgi-enriched lipids such as PtdIns4P. Due to the positively charged nature of PtdIns4P, it can interact with proteins containing negatively charged regions, such as NLRP3 and STUB1, thereby enabling Golgi PtdIns4P to regulate inflammation and protein degradation, respectively. The Golgi PtdIns4P library is generated by lipid kinases PI4KB, PI4K2A, and PI4K2B to produce PtdIns4P from PtdIns. Conversely, the lipid phosphatase SACM1L dephosphorylates PtdIns4P. SACM1L activity is regulated by alterations in its distribution in the ER and Golgi apparatus. The enrichment of SACM1L in the latter compartment is promoted by glucose or serum deprivation, leading to a decrease in PtdIns4P levels in the Golgi apparatus. This may affect pleiotropic intracellular processes through PtdIns4P interactors.

[0006] To date, hormone signaling remains the best-documented mechanism for coordinating glucose availability and systemic lipolysis. In the context of this invention, a general intrinsic cellular mechanism for balancing glucose and FA levels by regulating lipolysis via intracellular glucose sensing was investigated. Interestingly, a novel regulatory mechanism involving the PtdIns4P ligase complex CUL7 in the Golgi apparatus was identified. FBXW8 It plays a central role in the assembly of E3 ligase complexes, which in turn polyubiquitinate ATGL for proteasome-targeted degradation. Notably, intracellular glucose depletion is sensed through this pathway, leading to reduced assembly of the E3 ligase complex and increased ATGL-driven lipolysis. In this way, the glucose and FA libraries are intertwined.

[0007] Based on the aforementioned prior art, the object of the present invention is to provide means and methods for ex vivo fat reduction of fatty liver. This object is achieved through the subject matter of the independent claims of this specification and the further advantageous embodiments described in the dependent claims, embodiments, drawings, and general description. Summary of the Invention

[0008] A first aspect of the present invention relates to a method for ex vivo fat reduction of fatty liver, the method comprising: The fatty liver graft is perfused using a perfusion solution in the perfusion step, said perfusion solution comprising a compound selected from PI4KB inhibitors of formulas 1, 2, and 3, or a pharmaceutically acceptable salt or solvate thereof. (1) (2) (3), in - Z can be (methoxy)(methyl)phenyl, (methyl)(trifluoromethoxy)phenyl, (methoxy)(methyl)pyridyl, (ethyl)(methoxy)pyridyl, (ethoxy)(methyl)pyridyl, or dimethoxypyridyl. - A 11 It can be H, methyl, ethyl, hydroxymethyl, or hydroxyethyl. - Q is a heterocyclic ring as shown in Equation 4. (4), in X is N or C(H), Y is N or C(H), T is either S or N(CH3) - R 2 R 4 and R 5 Independently selected from H, methyl, and ethyl, - R 6a SO2R 8 , where R 6a In opposite or between positions - R 7a Selected from OH, OCH3, halogens, NH2, CH2-OH and NH-CO-R 8 , - Each R 7b Independently selected from OH, halogen, CN, NO2, C1-C6 alkyl, C1-C6 alkoxy, OCF3, OCH2CF3, OCH2Ph, OCH2-cyclopropyl, OCH2CH2OH, OCH2CH2NMe2, CF3, OCF3, C1-C6 alkyl-OH, NHSO2R 9 , NHCOR 8 and NR 9R 10 , - c is 0 or 1 in R 8 C1-C6 alkyl or NR 9 R 10 R 9 and R 10 Independently selected from H and C1-C6 alkyl groups.

[0009] This invention utilizes an intrinsic cellular mechanism involving Golgi PtdIns4P-mediated regulation of lipolysis driven by intracellular glucose-sensing triglyceride lipase (ATGL) (see...). Figure 5 f). Mechanistically, intracellular glucose depletion leads to lower Golgi PtdIns4P levels, which in turn reduces the E3 ligase complex CUL7. FBXW8 Assembly in the Golgi apparatus is necessary because the assembly of the E3 ligase complex requires the presence of PtdIns4P. Decreased levels of the E3 ligase complex lead to reduced polyubiquitination of ATGL in the Golgi apparatus, thereby decreasing proteasome-mediated degradation. Consequently, ATGL levels increase, and ATGL-driven lipolysis is enhanced. This intrinsic cellular mechanism regulates the intracellular fatty acid (FA) pool and their extracellular release to meet physiological needs, including fasting and glucose deprivation.

[0010] The enhanced ATGL-driven lipolysis was observed not only after intracellular glucose depletion but also through inhibition of the lipid kinase PI4KB, which generates PtdIns4P from PtdIns. The compound for which protection is sought is a PI4KB inhibitor that can be used for pharmacological manipulation of the Golgi apparatus PtdIns4P-CUL7. FBXW8 -ATGL axis, as illustrated in mouse models of simple hepatic steatosis and NASH. Administration of the claimed PI4KB inhibitor resulted in therapeutic improvement, i.e., delipidemia of the fatty liver in mice. Similarly, delipidemia of human fatty liver grafts was achieved by using ex vivo perfusion of the claimed PI4KB inhibitor.

[0011] Terms and Definitions

[0012] General

[0013] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any definition set forth below and any reference incorporated herein by reference, the definition set forth herein shall prevail.

[0014] As used herein, the terms “comprising,” “having,” “containing,” and “including,” as well as other similar forms and their grammatical equivalents, are intended to be equivalent in meaning and are open-ended, because one or more items following any of these words do not imply an exhaustive list of those items or that they are limited to only the listed items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only), or may contain not only components A, B, and C, but may also include one or more other components. Therefore, it is intended and understood that “comprising” and its similar forms and their grammatical equivalents encompass disclosures of embodiments “consistently consisting of” or “comprises of.”

[0015] Where numerical ranges are provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value of one-tenth of the unit of the lower limit between the upper and lower limits of the range, and any other stated value or intermediate value within the range, is covered by the invention but is subject to any specific exclusions within the range. Where the range contains one or two limits, the range excluding any one or two of those included limits is also included in this disclosure.

[0016] The “about” values ​​or parameters mentioned in this article include (and describe) changes involving that value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0017] As used herein, the singular forms “a,” “or,” and “the” included in the appended claims contain plural references unless the context clearly indicates otherwise.

[0018] "And / or" is used here as a specific description of each of two particular features or components, with or without other features or components. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to encompass "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley and Sons, Inc.) as well as chemical methods.

[0020] Specific terms

[0021] The term "fatty liver disease" refers to a condition in which excessive fat accumulates in the liver. Synonyms include "hepatic steatosis" and "slow liver disease" (SLD).

[0022] In the context of this specification, the term fatty liver disease encompasses non-inflammatory hepatic steatosis (also known as "simple hepatic steatosis") as well as inflammatory hepatic steatosis (steatohepatitis).

[0023] Hepatic steatosis is caused by damage to fatty acid metabolism, leading to the accumulation of triglyceride fats in the cytoplasm. It is diagnosed when fat in the liver exceeds 5–10% of its weight.

[0024] Initially, hepatocytes have liposomes surrounding the nucleus without replacing the centrally located nucleus (microvesicle steatosis). In later stages, the liposomes increase in size, and the nucleus shifts peripherally, producing a characteristic stamp-ring appearance (macrovesicle steatosis). Macrovesicle steatosis is the most common form and is often associated with alcohol, diabetes, obesity, and corticosteroids. Acute fatty liver of pregnancy and Reye's syndrome are examples of hepatic steatosis caused by microvesicle steatosis.

[0025] Severe fatty liver is sometimes accompanied by inflammation, a condition known as steatohepatitis. Progression to alcoholic steatohepatitis (ASH) or non-alcoholic steatohepatitis (NASH) depends on the persistence or severity of the underlying cause. The pathological damage in both conditions is similar. The onset of steatosis (lipid retention) and steatohepatitis can represent successive stages in the progression of fatty liver disease.

[0026] Based on different etiologies, fatty liver disease can be divided into the following subtypes (Renella et al. 2023, Hepatology 78(6):1966–1986, especially...). Figure 5 and Figure 6 ): - Fatty liver disease associated with metabolic dysfunction (MASLD). - Metabolic and alcoholic liver disease (MetALD). - Alcohol-related liver disease (ALD). - Cryptogenic fatty liver disease, - Fatty liver disease with specific etiologies.

[0027] In the context of this specification, terms such as MASLD, metALD, or ALD refer to non-inflammatory hepatic steatosis.

[0028] When accompanied by inflammation, MASLD can develop into metabolic dysfunction-associated steatohepatitis (MASH). Similarly, metALD and ALD can develop into metabolic and alcohol-related steatohepatitis and alcoholic steatohepatitis (ASH), respectively.

[0029] Cryptogenic fatty liver disease involves fatty liver disease without an identifiable cause. It can develop into cryptogenic steatohepatitis.

[0030] SLD with specific etiologies includes, for example, drug-induced or monogenic fatty liver disease. SLD with specific etiologies can develop into steatohepatitis with specific etiologies.

[0031] The terms “metabolic dysfunction-associated fatty liver disease” (MASLD) and “non-alcoholic fatty liver disease” (NAFLD) are used interchangeably.

[0032] The terms “metabolic dysfunction-associated steatohepatitis” (MASH) and “non-alcoholic steatohepatitis” (NASH) are used interchangeably.

[0033] In the context of this specification, the term "PI4KB" refers to phosphatidylinositol 4-kinase β, and more particularly to the proteins referred to by the UniProt identifiers Q9UBF8 (human PI4KB) and Q8BKC8 (mouse). Phosphatidylinositol 4-kinase β catalyzes the phosphorylation of phosphatidylinositol at the D-4 position, producing phosphatidylinositol-4-phosphate.

[0034] In the context of this specification, the term "PtdIns4P" refers to phosphatidylinositol-4-phosphate.

[0035] In the context of this specification, the term "PtdIns" refers to phosphatidylinositol.

[0036] In the context of this invention, the term "ATGL" refers to triglyceride lipase, and in particular to the protein referred to by the UniProt identifiers Q96AD5 (human) and Q8BJ56 (mouse).

[0037] The term "IGL-1 solution" refers to a solution having the following composition: Lactobionic acid: 35.8 g / L; Adenosine: 1.336 g / L; Allopurinol: 0.136 g / L; Glutathione: 0.922 g / L; Polyethylene glycol (PEG 35000): 1 g / L; Potassium dihydrogen phosphate: 3.402 g / L; Raffinose pentahydrate: 17.84 g / L; Magnesium sulfate heptahydrate: 1.232 g / L; Sodium hydroxide: sufficient; pH: 7.4; Water for injection: sufficient to 1 liter.

[0038] IGL-1 is a clear, sterile, pyrogen-free solution for the cryogenic flushing and storage of organs (kidney, liver, pancreas). The IGL-1 solution has an osmotic pressure of 290 mOsm / kg at room temperature, a total sodium concentration of 120 mmol / L, a total potassium concentration of 25 mmol / L, and a pH of 7.4.

[0039] The term "Belzer MPS" refers to a solution having the following composition:

[0040] HEPES (free acid) 2.38 g 10 mmol / L Potassium dihydrogen phosphate 3.4 g 25 mmol / L Mannitol (USP) 5.4 g 30 mmol / L Glucose (anhydrous), βD (+) 1.80 g 10 mmol / L Sodium gluconate 17.45 g 80 mmol / L Anhydrous magnesium gluconate 1.13 g 5 mmol / L Ribose, D(-) 0.75 g 5 mmol / L Pentafraction (HES) 50.0 g 50 g / L Glutathione (reduced form) 0.92 g 3 mmol / L Adenine (free base) 0.68 g 5 mmol / L Sodium hydroxide Sufficient quantity, pH: 7.4 Water for Injection Add enough to 1000 mL

[0041] Belzer MPS is a clear to pale yellow, sterile, pyrogen-free, and non-toxic solution for in vitro flushing and temporary continuous perfusion preservation of explant organs. This solution has a calculated osmolarity of approximately 300 mOsm / kg, a potassium concentration of 25 mmol / L, a sodium concentration of 100 mmol / L, and a pH of approximately 7.4 at room temperature.

[0042] The term "Belzer UW solution" (University of Wisconsin solution) refers to a solution having the following composition:

[0043] Lactobionic acid (as a lactone) 35.83 g 105 mmol / L Potassium dihydrogen phosphate 3.40 g 25 mmol / L Magnesium sulfate heptahydrate 1.23 g 5 mmol / L Five-hydrate raffinose 17.83 g 30 mmol / L adenosine 1.34 g 5 mmol / L Allopurinol 0.136 g 1 mmol / L Total glutathione 0.922 g 3 mmol / L potassium hydroxide 5.61 g 100 mmol / L Sodium hydroxide / hydrochloric acid Sufficient quantity, pH: 7.4 Water for Injection Add enough to 1000 mL

[0044] The term "Williams E" refers to the cell culture medium described in Williams, GM, and Gunn, JM, Long-Term Cell Culture of Adult Rat Liver Epithelial Cells. Exp. Cell Research, 89, 139-142 (1974) and Williams, GM et al., Isolation and Long-Term Cell Culture of Epithelial-Like Cells From Rat Liver. Exp. Cell Research, 69, 106-112 (1971).

[0045] The term “DMEM” refers to the cell culture medium described in Dulbecco, R., and Freeman, G., Plaque Production by the Polyoma Virus. Virology, 8, 396-397 (1959); Smith, JD, Freeman, G., Vogt, M., and Dulbecco, R., The Nucleic Acid of Polyoma. Virus, 12, 185-196 (1960); Morton, HJ, A Survey of Commercially Available Tissue Culture Media. InVitro, 6, 89 (1970); Rutzky, LP, and Pumper, RW, Supplement to a Survey of Commercially Available Tissue Culture Media (1970). In Vitro, 9, 468 (1974).

[0046] General molecular biology: nucleic acid sequence, expression

[0047] The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a specific polypeptide or protein after transcription and translation. Polynucleotide sequences can be used to identify larger segments or full-length coding sequences of genes associated with them. Methods for isolating larger sequence segments are known to those skilled in the art.

[0048] In the context of this specification, the term "recombinant" refers to nucleic acids, which are the product of one or more steps of cloning, restriction, and / or ligation, and which differ from naturally occurring nucleic acids. Recombinant viral particles include recombinant nucleic acids.

[0049] The term “gene expression” or “expression,” or the term “gene product,” can refer to one or both of the processes—or products—of the production of nucleic acids (RNA) or peptides or polypeptides (also referred to as transcription and translation, respectively), or the processing of genetic information to produce polypeptide products. The term “gene expression” can also be applied to RNA gene products, such as the transcription and processing of regulatory RNA or structural (e.g., ribosomal) RNA. If the expressed polynucleotide is derived from genomic DNA, expression can be included in the splicing of mRNA in eukaryotic cells. Expression can be measured at the transcriptional and translational levels, in other words, the mRNA and / or protein products.

[0050] inhibition

[0051] In the context of this specification, the term "inhibitor" refers to any pharmaceutically acceptable agent or compound that can interact with its designated target (phosphatidylinositol 4-kinase, particularly PI4KB in the case of this specification) and specifically interfere with the biological activity of its designated target. Inhibitors comprise small molecule drugs that satisfy Lipinski's five rules (the drug must satisfy at least three of the following rules: number of hydrogen bond donors ≤ 5; number of hydrogen bond acceptors ≤ 10; molecular weight < 500 Da; octanol / water partition coefficient ≤ 5). Specific examples of such inhibitors are mentioned herein.

[0052] In the context of this invention, the term "PI4KBK inhibitor" refers to compounds that can be used to interfere with the biological activity of phosphatidylinositol 4-kinase β (PI4KB), i.e., phosphatidylinositol phosphorylation to produce phosphatidylinositol-4-phosphate. The term "PI4KBK inhibitor" includes compounds of formulas 1, 2, and 3 as claimed. Specific examples of PI4KB inhibitors are UCB9608, BF738735, and PIK93. (UCB9608) (BF738735) (PIK93).

[0053] treat

[0054] As used herein, the term "treatment" for any disease or condition (e.g., NAFLD or NASH) in one embodiment refers to improving the disease or condition (e.g., slowing or halting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" refers to reducing or improving at least one bodily parameter, including those that the patient may not be able to discern. In yet another embodiment, "treatment" refers to regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or both. Unless specifically described below, methods for assessing the treatment and / or prevention of disease are generally known in the art. In the context of this invention, the treatment of fatty liver disease specifically relates to reducing hepatic triglyceride levels and / or preventing the progression of NASH caused by simple hepatic steatosis.

[0055] Organic Chemistry

[0056] The chemical formulas in this specification follow organic chemistry conventions and do not show hydrogen atoms on the carbon skeleton. Carbon is tetravalent, and unless otherwise indicated, any bonds not shown are assumed to be hydrogen. Hydrogen can be exchanged with deuterium without altering the bulk chemical properties of the molecule; however, in the case of dye or pharmaceutical molecules, the exchange of hydrogen with deuterium may result in changes in the spectral properties of the molecule or its acceptor interactions. Unless otherwise expressly stated herein, the disclosure of chemical formulas explicitly or implicitly shown by convention restated in the first sentence of this paragraph covers molecules in which one or more hydrogen atoms are exchanged with deuterium.

[0057] The term "(methoxy)(methyl)phenyl" refers to a phenyl moiety substituted with both methoxy (-OCH3) and methyl (-CH3) substituents. Similarly, "(methyl)(trifluoromethoxy)phenyl" refers to a phenyl moiety substituted with both methyl and trifluoromethoxy (-O-CF3) substituents.

[0058] The term "(methoxy)(methyl)pyridyl" refers to a pyridyl moiety substituted with a methoxy (-OCH3) substituent and a methyl (-CH3) substituent. Similarly, "(ethyl)(methoxy)pyridyl", "(ethoxy)(methyl)pyridyl" and "dimethoxypyridyl" refer to a pyridyl moiety substituted with an ethyl (-CH2-CH3), methoxy (-OCH3), ethoxy (-O-CH2-CH3), methyl (-CH3), or both methoxy (-OCH3) substituents, respectively.

[0059] The terms “hydroxymethyl” and “hydroxyethyl” refer to -CH2-OH and -CH2-CH2-OH, respectively.

[0060] The term "halogen" refers to one of fluorine, chlorine, bromine, and iodine. In certain embodiments, this term refers to a member of the group consisting of chlorine, bromine, and iodine. In other specific embodiments, this term refers to a member of the group consisting of chlorine and bromine.

[0061] In the context of this specification, the term "alkyl" refers to saturated straight-chain, branched, or (partially or fully) cyclic hydrocarbons. In particular, the term "alkyl" refers to saturated straight-chain hydrocarbons.

[0062] In the context of this specification, the term "C1-C4 alkyl" refers to a saturated straight-chain or branched hydrocarbon having 1, 2, 3, or 4 carbon atoms. Non-limiting examples of C1-C4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, cyclobutyl, cyclopropyl, and methylcyclopropyl. In some embodiments, the C1-C4 alkyl is a methyl, ethyl, propyl, or butyl moiety.

[0063] In the context of this specification, "C1-C6 alkyl" refers to a saturated straight-chain or branched hydrocarbon having 1, 2, 3, 4, 5, or 6 carbon atoms. Non-limiting examples of C1-C6 alkyl include the examples of C1-C4 alkyl given above, and also n-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2-dimethylpropyl, cyclopentyl, cyclohexyl, and methylcyclopentyl. In some embodiments, the C5 alkyl group is an pentyl or cyclopentyl moiety and the C6 alkyl group is a hexyl or cyclohexyl moiety.

[0064] When used in the context of a chemical formula, the following abbreviations may be used: "Me" for methyl CH3, "Et" for ethyl-CH2CH3, and "Prop" for propyl-(CH2)2CH3 (n-propyl). n -pr) or -CH(CH3)2 (isopropyl, i -pr), "but" is butyl-C4H9, -(CH2)3CH3, -CHCH3CH2CH3, -CH2CH(CH3)2 or -C(CH3)3.

[0065] As used herein, the term "pharmaceutical composition" refers to the compound of the present invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions according to the present invention are provided in a form suitable for topical, parenteral, or injectable administration.

[0066] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic agent, absorption delay agent, salt, preservative, pharmaceutical, pharmaceutical stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, and combinations thereof (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). This invention also includes nanoparticles, liposomes, or cell carriers within the meaning of "pharmaceutically acceptable carrier." Detailed Implementation

[0067] A first aspect of the present invention relates to a method for ex vivo fat reduction of fatty liver, the method comprising: The fatty liver graft is perfused using a perfusion solution in the perfusion step, said perfusion solution comprising a compound selected from PI4KB inhibitors of formulas 1, 2, and 3, or a pharmaceutically acceptable salt or solvate thereof. (1) (2) (3), in - Z can be (methoxy)(methyl)phenyl, (methyl)(trifluoromethoxy)phenyl, (methoxy)(methyl)pyridyl, (ethyl)(methoxy)pyridyl, (ethoxy)(methyl)pyridyl, or dimethoxypyridyl. - A 11 It can be H, methyl, ethyl, hydroxymethyl, or hydroxyethyl. - Q is a heterocyclic ring as shown in Equation 4. (4), in X is N or C(H), Y is N or C(H), T is either S or N(CH3) - R 2 R 4 and R 5 Independently selected from H, methyl, and ethyl, - R 6a SO2R 8 , where R 6a In opposite or between positions - R 7a Selected from OH, OCH3, halogens, NH2, CH2-OH and NH-CO-R 8 , - Each R 7bIndependently selected from OH, halogen, CN, NO2, C1-C6 alkyl, C1-C6 alkoxy, OCF3, OCH2CF3, OCH2Ph, OCH2-cyclopropyl, OCH2CH2OH, OCH2CH2NMe2, CF3, OCF3, C1-C6 alkyl-OH, NHSO2R 9 , NHCOR 8 and NR 9 R 10 , - c is 0 or 1 in R 8 C1-C6 alkyl or NR 9 R 10 R 9 and R 10 Independently selected from H and C1-C6 alkyl groups.

[0068] Another embodiment of the present invention relates to a method for ex vivo fat reduction of fatty liver, the method comprising perfusing a fatty liver graft with a perfusion solution in a perfusion step, the perfusion solution comprising a PI4KB inhibitor or a pharmaceutically acceptable salt or solvate thereof.

[0069] The term "(methoxy)(methyl)phenyl" refers to a phenyl moiety substituted with both methoxy (-OCH3) and methyl (-CH3) substituents. Similarly, "(methyl)(trifluoromethoxy)phenyl" refers to a phenyl moiety substituted with both methyl and trifluoromethoxy (-O-CF3) substituents.

[0070] The term "(methoxy)(methyl)pyridyl" refers to a pyridyl moiety substituted with a methoxy (-OCH3) substituent and a methyl (-CH3) substituent. Similarly, "(ethyl)(methoxy)pyridyl", "(ethoxy)(methyl)pyridyl" and "dimethoxypyridyl" refer to a pyridyl moiety substituted with an ethyl (-CH2-CH3), methoxy (-OCH3), ethoxy (-O-CH2-CH3), methyl (-CH3), or both methoxy (-OCH3) substituents, respectively.

[0071] In some embodiments, the compound is selected from PI4KB inhibitors of formulas 1, 2 and 3, or pharmaceutically acceptable salts or solvates thereof.

[0072] In some embodiments, the compound is selected from PI4KB inhibitors of formulas 1, 2 and 3.

[0073] In some embodiments, the compound is a PI4KB inhibitor of Formula 1.

[0074] In some implementations, the fatty liver graft is a human fatty liver graft.

[0075] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is from 0.1 to 100 µM.

[0076] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 1 to 100 µM.

[0077] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is from 0.1 to 40 µM.

[0078] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 1 to 40 µM.

[0079] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 5 to 40 µM.

[0080] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 10 to 40 µM.

[0081] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is from 0.1 to 20 µM.

[0082] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 1 to 20 µM.

[0083] In some embodiments, the concentration of the PI4KB inhibitor in the perfusion fluid is 10 to 20 µM.

[0084] To reach the therapeutic window, the initial concentration of the PI4KB inhibitor may be higher than the target concentration during subsequent perfusion. Typically, the perfusion fluid undergoes hemodialysis during perfusion. To address losses due to hemodialysis, some of the PI4KB inhibitor (and other added compounds) is washed away over time, and additional boluses are added daily to maintain its position within the therapeutic window. Depending on how the PIK4B inhibitor (or another added compound) dissolves, it can also be administered via continuous infusion using an infusion pump.

[0085] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is from 0.1 to 100 µM.

[0086] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 1 to 100 µM.

[0087] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 10 to 100 µM.

[0088] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 0.1 to 40 µM.

[0089] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 1 to 40 µM.

[0090] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 10 to 40 µM.

[0091] In some embodiments, the initial concentration of the PI4KB inhibitor in the perfusion fluid is 20 µM.

[0092] In some embodiments, the concentration of the PI4KB inhibitor is controlled and adjusted during the perfusion step to a concentration of 0.1 to 20 µM in the perfusion solution.

[0093] In some embodiments, the concentration of the PI4KB inhibitor is controlled and adjusted during the perfusion step to a concentration of 1 to 20 µM in the perfusion solution.

[0094] In some embodiments, the concentration of the PI4KB inhibitor is controlled and adjusted during the perfusion step to a concentration of 5 to 20 µM in the perfusion solution.

[0095] In some embodiments, the concentration of the PI4KB inhibitor is controlled and adjusted during the perfusion step to a concentration of 10 µM in the perfusion solution.

[0096] PI4KB inhibitors, particularly UCB9608, can be added via bolus injection. UCB9608 dissolved in DMSO precipitates when mixed with water and is not recycled. Therefore, PI4KB inhibitors, especially UCB9608, can be added via bolus injection rather than continuous administration via a syringe pump to avoid precipitation within the syringe.

[0097] Dosage adjustment depends on the dialysis flow rate and dialysis filter. The clearance rate of the compound can be optimally measured using LC-MS for a specific dialysis filter and flow rate. The dosage can then be adjusted accordingly by administering a bolus or continuous infusion.

[0098] Typically, 1.5 to 3.5 L of perfusion fluid is used for perfusion. To achieve a concentration of 10 µM, 15 µmol of PI4KB inhibitor, particularly UCB9608, can be added to 1.5 L of perfusion fluid via bolus injection. For ease of handling, the PI4KB inhibitor can be dissolved in a suitable solvent, such as DMSO, before being added to the perfusion fluid.

[0099] If a PI4KB inhibitor, such as UCB9608, is water-insoluble, water solubility can be achieved through encapsulation of the PI4KB inhibitor or by forming a complex with albumin. Administration by encapsulation or as a albumin-bound complex offers the benefit of preventing elution in the perfusion circuit via the dialysis bypass, reducing the risk of precipitation in the perfusion reservoir, and potentially improving cellular uptake of the inhibitor.

[0100] In some implementations, the concentration of the PI4KB inhibitor is adjusted by the following - Add PI4KB inhibitor via bolus injection, wherein the PI4KB inhibitor is soluble in an organic solvent, particularly DMSO, or - Continuous application, wherein the PI4KB inhibitor is bound to albumin, or the PI4KB inhibitor is encapsulated in a nanoemulsion, or the PI4KB inhibitor is part of a nanoparticle.

[0101] In some implementations, the concentration of the PI4KB inhibitor is adjusted by adding it to a daily bolus injection.

[0102] In some implementations, the concentration of the PI4KB inhibitor is adjusted by adding 10 to 100 µmol per liter of perfusion solution via daily bolus injection.

[0103] In some implementations, the concentration of the PI4KB inhibitor is adjusted by adding 10 to 40 µmol per liter of perfusion solution via daily bolus injection.

[0104] In some implementations, the concentration of the PI4KB inhibitor is adjusted by adding 10 µmol of the daily bolus per liter of perfusion fluid.

[0105] In some implementations, the PI4KB inhibitor is dissolved in a suitable solvent.

[0106] In some implementations, the PI4KB inhibitor is dissolved in an organic solvent.

[0107] In some implementations, the PI4KB inhibitor is dissolved in DMSO (dimethyl sulfoxide).

[0108] In some implementations, the daily bolus injection of the PI4KB inhibitor is added dropwise.

[0109] In some implementations, the daily bolus of the PI4KB inhibitor is added dropwise over one minute.

[0110] For liver transplantation, perfusion is performed until a non-fatty liver is achieved. Liver viability and function are determined by a variety of perfusion fluid alternative markers and perfusion parameters known to those skilled in the art. Regarding organ defatting, puncture biopsy is the most reliable way to assess the effectiveness of treatment / fat reduction. Non-limiting examples of revealing lipid content in biopsies include measuring triglycerides in frozen tissue or performing histological evaluation with simple H&E staining or lipid-specific staining such as Nile Red or Oil Red O staining. This allows for reassessment of microcystic and macrocystic fatty degeneration in the tissue. Transplant centers have their own thresholds for microcystic and macrocystic fatty degeneration used for selecting transplant organs. In addition to invasive biopsy-related assessments, the weight of the entire organ can serve as an indicator of defatting. Furthermore, non-esterified fatty acids (NEFAs) can be measured in waste dialysate.

[0111] In some implementations, a perfusion step is performed until a non-fatty liver is obtained.

[0112] In some implementations, the infusion process is performed for 2 to 14 days.

[0113] In some implementations, the infusion process is performed over 6 to 10 days.

[0114] In some implementations, the infusion process is performed over 8 days.

[0115] Most perfusion protocols use leukocyte-depleted blood as the perfusion medium. Leukocyte-anemic blood can be obtained by separating leukocytes from whole blood. Suitable methods are known to those skilled in the art. For example, leukocytes can be separated from whole blood by affinity separation, freeze-thaw, centrifugation, or filtration, based on size and dielectric properties. Blood can be obtained from organ donors and used as whole blood or leukocyte-anemic blood.

[0116] Blood without white blood cells can be obtained by mixing red blood cells, fresh frozen plasma, and platelets. White blood cells are absent.

[0117] Alternatively, oxygenated blood substitutes can be used as perfusion media. Oxygenated blood substitutes can be obtained by mixing red blood cells with organ preservation solutions or cell culture media. Non-limiting examples of organ preservation solutions include MPS Belzer, IGL-1, and UW. Non-limiting examples of suitable cell culture media include Williams E or DMEM.

[0118] Synthetic oxygen carriers can be used, such as those derived from HbO2 therapeutics (bovine hemoglobin polymerized from glutaraldehyde) and Hemarina (from...). Arenicola marina It is a synthetic oxygen carrier of extracellular hemoglobin (the oxygen carrier of red blood cells) to replace red blood cells.

[0119] Typically, the perfusion medium should be compatible with the donor's blood type. If this is not possible, an alternative compatible blood type can be used. For example, a liver from a donor with blood type A+ can be perfused with a perfusion medium containing blood cells of blood type A+.

[0120] In some embodiments, the perfusion fluid includes a perfusion medium, wherein the perfusion medium is whole blood, leukocyte-anemic blood, leukocyte-free blood, or an oxygenated blood substitute.

[0121] In some embodiments, the perfusion fluid includes a perfusion medium, wherein the perfusion medium is leukocyte-anemic blood or leukocyte-free blood.

[0122] In some embodiments, the perfusion fluid includes a perfusion medium, wherein the perfusion medium is leukocyte-free blood. Leukocyte-free blood is specifically obtained by mixing 6 units of erythrocyte concentrate (e.g., 200 to 250 mL / unit), 5 units of fresh frozen blood cells (e.g., 200 mL / unit), 1 unit of platelets (e.g., 175 to 400 mL / unit), and 200 mL of 20% human albumin (CSL Behring).

[0123] After all the blood is preserved and mixed, the final hematocrit of the perfusion fluid is approximately 30%. In some embodiments, the hematocrit level of the perfusion fluid is within the physiological range, particularly between 25% and 35%, and more particularly 30%.

[0124] In some implementations, whole blood is the donor's own whole blood.

[0125] In some implementations, the leukocyte-anemic blood is donor autologous leukocyte-anemic blood.

[0126] In some implementations, oxygenated blood substitutes - It is an organ preservation solution or cell culture medium containing red blood cells, and / or - It is a solution containing extracellular hemoglobin or hemoglobin-based oxygen carrier (HBOC).

[0127] In some implementations, extracellular hemoglobin is derived from Arenicola marina Extracellular hemoglobin.

[0128] In some implementations, HBOC is hemoglobin polymerized from glutaraldehyde.

[0129] As described above, the perfusion fluid undergoes hemodialysis during perfusion to allow control and / or correction of parameters such as the concentration of PI4K inhibitors and other additives, pH, hematocrit levels, or waste removal.

[0130] In some implementations, the perfusion fluid undergoes dialysis during the perfusion step.

[0131] In some implementations, the perfusion solution undergoes dialysis during the perfusion step to control and / or correct the concentration of the PI4K inhibitor.

[0132] In some embodiments, the perfusion fluid undergoes dialysis during the perfusion step to... - Control and / or adjust the concentration of PI4K inhibitors. - Control and / or correct electrolyte concentration, - Control and / or correct pH, - Remove waste, - Maintain hematocrit levels within the physiological range, especially between 25% and 35%.

[0133] In some embodiments, the perfusion fluid undergoes dialysis during the perfusion step to... - Control and / or correct electrolyte concentration, - Control and / or correct pH, - Remove waste, - Maintain hematocrit levels within the physiological range, especially between 25% and 35%.

[0134] The perfusion fluid may also include additives such as insulin to regulate glucose concentration, vasodilators to maintain a constant flow of perfusion fluid through the blood vessels of the liver transplant, bile acids to stimulate bile, anticoagulants to prevent blood clotting, antibiotics to prevent bacterial growth, or glucocorticoids (which have anti-inflammatory and immunosuppressive effects).

[0135] In some embodiments, the perfusion fluid also includes one or more additives selected from insulin, vasodilators, bile acids, anticoagulants, antibiotics, and steroids.

[0136] Insulin can be administered at a concentration of 0.45 IU / mL. The infusion rate is automatically controlled by a controller based on real-time glucose measurements to maintain glucose concentrations between 3 and 9 mmol / L.

[0137] In some implementations, insulin is added to the perfusion fluid.

[0138] In some implementations, insulin is added to the perfusion fluid in an amount that maintains a glucose concentration of 3 to 9 mM, particularly 7 mM.

[0139] Vasodilators such as eprostol can be administered at a concentration of 4 mg / mL. Similar to insulin, the total amount of vasodilator added is automatically controlled by measuring the flow rate in the hepatic artery. When the flow rate drops below 0.25 L / min, the infusion is gradually increased until the flow rate stabilizes at 0.25 L / min.

[0140] In some embodiments, a vasodilator is added to the perfusion fluid.

[0141] In some embodiments, a vasodilator is added to the perfusion solution at a flow rate of 0.25 L / min in the hepatic artery.

[0142] In some embodiments, the vasodilator is eprostol.

[0143] In some implementations, antibiotics are added to the infusion fluid.

[0144] In some embodiments, 1.5 to 3.5 g, particularly 2.25 g, of antibiotics are added to the perfusion solution every 24 hours for every 2500 ml of perfusion solution during the perfusion step.

[0145] In some implementations, the antibiotic is tazobactam.

[0146] In some embodiments, steroids are added to the perfusion fluid.

[0147] In some embodiments, during the perfusion step, a bolus of 300 to 1000 mg, particularly 0.5 g, of steroid is added every 24 hours for every 2500 ml of perfusion solution.

[0148] In some implementations, the steroid is a glucocorticoid.

[0149] In some implementations, the steroid is methylprednisolone.

[0150] In some embodiments, bile acids are added to the perfusion fluid.

[0151] In some embodiments, bile acids are added at a rate of 50 to 100 mg / h, particularly 70 mg / h, per 2500 ml of perfusion solution during the perfusion step.

[0152] In some embodiments, the bile acid is ursodeoxycholic acid or taurocholic acid.

[0153] In some implementations, an anticoagulant is added to the perfusion fluid.

[0154] In some implementations, 1,000 to 1,500 U, particularly 1,000 U, of anticoagulant is added per hour during the infusion step.

[0155] In some implementations, the anticoagulant is heparin.

[0156] At the start of the infusion process, the additive may be added discontinuously, with the initial concentration achieved by adding an initial bolus to the infusion medium.

[0157] In some embodiments, the infusion medium includes - 1.5 to 3.5 g of antibiotic per 2500 ml of perfusion medium, especially the initial bolus of 2.25 g of antibiotic, and / or - 300 mg to 1000 mg per 2500 ml perfusion medium, especially the first bolus of 0.5 g steroid.

[0158] In some embodiments, the infusion medium includes - 1.5 to 3.5 g per 2500 ml of perfusion medium, especially the first bolus of 2.25 g tazobactam, and / or - 300 mg to 1000 mg per 2500 ml perfusion medium, especially the first bolus of 0.5 g methylprednisolone.

[0159] Mammalian enzymes, including liver enzymes such as ATGL, typically achieve their maximum activity under normothermic conditions, i.e., at a temperature of approximately 37°C. Therefore, it is beneficial to perform the perfusion procedure under normothermic conditions. The perfusion procedure can therefore also be referred to as the normothermic perfusion procedure.

[0160] In some implementations, the infusion step is carried out at 34 to 37°C, particularly at 37°C.

[0161] In some implementations, the infusion is performed under the following conditions - Hepatic artery pressure of 70–90 mmHg systolic and 40–60 mmHg diastolic (mean arterial flow of 0.25 L / min). - Portal vein pressure of 2–10 mmHg (mean portal vein flow of 1 L / min). - 12 kPa oxygen partial pressure, >98% oxygen saturation - Oxygen saturation of 65% in the vena cava. - Constant pressure of 0 mmHg in the vena cava.

[0162] In some embodiments, the method further includes a preparation step, the preparation step comprising... - Close the inferior vena cava, especially by suturing, and / or - Intubate the hepatic artery, portal vein, superior hepatic vena cava, and common bile duct.

[0163] Prior to normothermic perfusion as described herein, the liver may be flushed to cool the organ and thus slow metabolism. Secondly, blood cells should be flushed and removed to prevent clotting problems. The flushing step is typically performed in every standard transplant procedure. For example, it can be performed not only during immediate liver perfusion as described herein, but also when the liver is not immediately connected to a normothermic perfusion machine but is first transported to another hospital where normothermic perfusion and subsequent liver transplantation are performed.

[0164] In contrast to perfusion, during perfusion the perfusion fluid circulates through the liver graft several times, while during the flushing step the solution only passes through the organ once (i.e., flushing).

[0165] The first cold rinse can be performed in situ, i.e., in situ cold rinse, sometimes followed by a second, non-in situ cold rinse. In situ cold rinse involves rinsing while the liver is still inside the donor organism (post-mortem). Non-in situ cold rinse involves rinsing outside the donor organism after the liver has been harvested from the (dead) donor.

[0166] In some embodiments, the method further includes a rinsing step, which includes rinsing the fatty liver graft with a preservation solution, particularly a preservation solution selected from IGL-1 solution, UW solution, and Belzer solution.

[0167] In some implementations, the flushing step is performed before the infusion step.

[0168] In some embodiments, the rinsing step includes in-situ cold rinsing, wherein the fatty liver graft is rinsed with an in-situ preservation solution.

[0169] In some implementations, the rinsing step includes ectopic cold rinsing, wherein the fatty liver graft is ectopically rinsed with a preservation solution.

[0170] In some embodiments, the rinsing step includes in-situ cold rinsing, wherein the fatty liver graft is rinsed in situ with a preservation solution, followed by ectopic cold rinsing, wherein the fatty liver graft is rinsed ectopically with a preservation solution.

[0171] In some embodiments, the preservation solution is selected from IGL-1 solution, UW solution, and Belzer solution.

[0172] After organ acquisition and in situ and / or ectopic cold flushing, the graft can be stored on ice, i.e., static cryopreservation (SCS), until it is connected to a perfusion device at normal body temperature.

[0173] In some embodiments, the method further includes a static refrigeration step, wherein the liver is stored on ice.

[0174] In some implementations, the static refrigeration step is performed after the rinsing step and before the filling step.

[0175] Performing hypothermic oxygenated perfusion, rather than static cryopreservation, may be beneficial for treatment before carrying out the perfusion steps described above, i.e., normothermic perfusion. Hypothermic oxygenated perfusion has a protective effect against ischemia-reperfusion injury to the graft. Therefore, hypothermic oxygenated perfusion therapy can be beneficial in maintaining the organ in better shape before starting actual treatment on a normothermic perfusion machine.

[0176] In some embodiments, the method further includes a cryogenic oxygen perfusion step, which includes perfusing the fatty liver graft with a cryogenic machine perfusion solution.

[0177] In some implementations, the cryogenic oxygenation perfusion step is performed after the flushing step and before the perfusion step.

[0178] In some implementations, the cryogenic machine perfusion solution is a Belzer MPS solution.

[0179] During the cryogenic oxygenation perfusion step, oxygen is introduced through a membrane oxygenator to achieve an oxygen partial pressure >100 kPa. The oxygen saturation of the perfusion solution is 100%. The oxygen flow rate to the oxygenator is at least 0.5 L / min, particularly 0.5 to 1 L / min.

[0180] In some implementations, the cryogenic oxygenation perfusion step is performed at an oxygen partial pressure of >100 kPa.

[0181] Cryogenic oxygen perfusion is usually performed on ice, ideally at 4°C.

[0182] In some implementations, the cryogenic oxygenation perfusion step is carried out at 0 to 12°C, particularly 8°C.

[0183] In some implementations, the cryogenic oxygenation perfusion step is performed at 4 to 8°C, particularly at 4°C.

[0184] In some implementations, the cryogenic oxygenation perfusion step is performed for 1 to 8 hours, particularly 2 hours.

[0185] In some embodiments, the compound is selected from PI4KB inhibitors of formulas 1a, 2a, and 3. (1a) (2a), Among them, Z and A 11 Q, R 2 R 4 R 5 R 6a R 7a R 7b c is as defined above.

[0186] In some embodiments, the compound is a PI4KB inhibitor of formula 1a.

[0187] In some implementations, A 11 It can be methyl, ethyl, hydroxymethyl, or hydroxyethyl.

[0188] In some implementations, A 11 It can be methyl or ethyl.

[0189] In some implementations, A 11 It is a methyl group.

[0190] In some implementations, A 11 It can be H, methyl, or ethyl.

[0191] In some implementations, A 11 It is H or methyl.

[0192] In some implementations, Z is selected from the portions of formulas Za, Zb, Zc, Zd, Ze, Zf, Zg, Zh, and Zi. (Za) (Zb) (Zc) (Zd) (Ze) Z(f), (Zg) (Zh) (Zi).

[0193] In some implementations, Z is selected from the portions of formulas Za, Zc, and Zd.

[0194] In some implementations, Z is a part of the formula Za.

[0195] In some implementations, one of X and Y is N and the other is C (H).

[0196] In some implementations, X is C(H) and Y is N.

[0197] In some implementations, Q is selected from heterocycles of formulas Qa, Qb, Qc, Qd, and Qe. (Qa) (Qb) (Qc) (Qd) (Qe).

[0198] In some implementations, Q is selected from heterocycles of formulas Qa and Qb.

[0199] In some implementations, Q is a heterocycle of the formula Qa.

[0200] In some implementations, A 11 It is a methyl group, and Q is selected from heterocycles of formulas Qa and Qb.

[0201] In some implementations, A 11 It is a methyl group, and Q is a heterocycle of formula Qa.

[0202] In some implementations, Q is selected from heterocycles of formulas Qa and Qb, and Z is selected from portions of formulas Za, Zc, and Zd.

[0203] In some implementations, Q is a heterocycle of formula Qa, and Z is selected from portions of formulas Za, Zc, and Zd.

[0204] In some implementations, A 11 Q is a methyl group, Q is selected from the heterocycles of formula Qa and Qb, and Z is selected from the parts of formulas Za, Zc and Zd.

[0205] In some implementations, A 11 Q is a methyl group, Q is a heterocycle of formula Qa, and Z is selected from the parts of formulas Za, Zc, and Zd.

[0206] In some implementations, Q is selected from heterocycles of formulas Qa and Qb, and Z is a part of formula Za.

[0207] In some implementations, Q is a heterocycle of formula Qa, and Z is a part of formula Za.

[0208] In some implementations, A 11 Q is a methyl group, Q is selected from heterocycles of formula Qa and Qb, and Z is a part of formula Za.

[0209] In some implementations, A 11 Q is a methyl group, Q is a heterocycle of formula Qa, and Z is a part of formula Za.

[0210] In some implementations, R 2 R 4 and R 5 It is independently selected from H or methyl.

[0211] In some implementations, R 2 It is methyl, and R 4 and R 5 For H.

[0212] In some implementations, R 6a SO2R 8 , where R 8 It is a C1-C6 alkyl group.

[0213] In some implementations, R 6a SO2R 8 , where R 8 It is CH3.

[0214] In some implementations, R 7a Selected from OH, NH2, and OCH3.

[0215] In some implementations, c is 1.

[0216] In some implementations, R 8 R9 and R9 are independently selected from C1-C6 alkyl groups.

[0217] In some implementations, R 8 and R 9 It is a methyl group.

[0218] In some implementations, R 7b Selected from OH, halogen, NO2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl-OH, NH2, NHSO2R 9 and NHCOR 8 , where R 9 and R 8 As defined above.

[0219] In some implementations, R 7b Selected from OH, halogen, NO2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl-OH, NH2, NHSO2R 9 and NHCOR 8 , where R 9 and R 8 It is independently selected from C1-C6 alkyl groups.

[0220] In some implementations, R 7b Selected from OH, halogen, NO2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl-OH, NH2, NHSO2R 9 and NHCOR 8 , where R 9 and R 8 It is a methyl group.

[0221] In some implementations, R 7b Selected from OH, halogens, NO2, OCH3, CF3, OCF3, CH3, CH2-OH and NHSO2R 8 , where R 8 As defined above.

[0222] In some implementations, R 7b Selected from OH, halogens, NO2, OCH3, CF3, OCF3, CH3, CH2-OH and NHSO2R 8 , where R 8 It is a C1-C6 alkyl group.

[0223] In some implementations, R 7b Selected from OH, halogens, NO2, OCH3, CF3, OCF3, CH3, CH2-OH and NHSO2R8 , where R 8 It is a methyl group.

[0224] In some implementations, R7b is a halogen.

[0225] In some implementations, R 7b It is -F.

[0226] In some embodiments, the compound is selected from PI4KB inhibitors of formulas 1b, 2b, and 3. (1b) (2b).

[0227] In some embodiments, the PI4KB inhibitor is a compound of formula 1b.

[0228] The invention is further illustrated by the following embodiments and accompanying drawings, from which further implementation methods and advantages can be derived. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0229] Figure 1: This study shows that glucose deprivation increases lipolysis by stabilizing ATGL in adipose tissue and liver. (a) Plasma FFA levels in wild-type mice after 24 hours of fasting (n=6 in both groups). (b) Quantification of ATGL levels in lysates from liver and adipose tissue of fed and fasted mice (n=6 in both groups). (c) Plasma FFA levels in wild-type mice 1 h, 6 h, and 12 h after administration of 2-DG (2 g / kg BW) (1 h and 6 h, n=6 for saline group, n=8 for 2-DG group; 12 h, n=10 for saline group, n=13 for 2-DG group). (d) Quantification of ATGL levels in lysates of liver and adipose reservoirs collected 1 h, 6 h, and 12 h after administration of 2-DG (2 g / kg BW) (1 h and 6 h, n=6 for saline group, n=8 for 2-DG group; 12 h, n=12 for saline group, n=15 for 2-DG group). (e) Plasma FFA levels in wild-type mice 5.5 h after treatment with 2-DG (2 g / kg BW) followed by administration of insulin (0.75 U / kg BW) 30 min (n=8 for all groups). (f) Plasma FFA levels in wild-type mice 6 h after co-administration of 2-DG (2 g / kg BW) and insulin (0.75 U / kg BW) (n=8 for all groups). (g) Quantification of ATGL and HSL Ser660ph levels in subcutaneous adipose tissue before and after EHC from insulin-sensitive patients (n=16 patients). (h) Quantitative levels of ATGL and HSL Ser660ph in subcutaneous adipose tissue before and after EHC in patients with insulin resistance (n=8 patients). Data are expressed as mean ± sem and analyzed using two-tailed unpaired t-tests (a, b, c, and d (1h in liver and iBAT, 1h and 6h in iWAT, and 1h and 6h in gWAT)), paired t-tests (h), Mann-Whitney test (d (6h and 12h in liver, 12h in iWAT, and 12h in gWAT)), Wilcoxon test (g), and ANOVA with Tukey correction (e and f).

[0230] Figure 2: This illustrates how glucose deprivation regulates lipolysis by stabilizing ATGL in the Golgi apparatus. (a) Quantification of ATGL in HepG2 cell types after 6 hours of culture at different glucose concentrations (based on Western blotting) (n=6 for all groups). (b) Quantification of ATGL in HepG2 cell types treated with 50 mM 2-DG and harvested at the indicated interval (n=9 for controls, 3 for all 2-DG groups) by Western blotting. (c) Quantification of ATGL in iBAs after 24 hours of culture at different glucose concentrations (by Western blotting) (n=6 for all groups). (d) Levels of non-esterified fatty acids (NEFA) released by basal iBAs in starved medium 24 hours after treatment with mediums of different glucose concentrations (n=6 for all groups). (e) Quantification of ATGL in iBAs after 24 hours of treatment with 2-DG at the indicated concentration (by Western blotting) (n=4 for all groups). (f) NEFA levels released by basal iBAs in starved medium 24 hours after treatment with 100 mM 2-DG (n=5 in both groups). (g) Quantification of ATGL in HEK293T cell types 6 hours after treatment with glucose-free medium (by immunoblotting) (n=4 in both groups). (h) Quantification of ATGL in HEK293T cell types 12 hours after treatment with 50 mM 2-DG (by immunoblotting) (n=4 in both groups). (i) Quantification of ATGL in HEK293-AAV cell types by immunofluorescence 6 hours after treatment with glucose-free medium (43 cells in both groups). (j) Quantification of ATGL in whole-cell extract (WCE), Golgi fraction, and vesicle fraction extracted from HEK293T cell types 6 hours after treatment with glucose-free medium (by immunoblotting) (n=6 in both groups). Results are presented as mean ± sem and analyzed using two-tailed unpaired t-tests (f to i), paired t-tests (j), ANOVA with Dunnett correction (for multiple comparisons between the control group and other groups) (a, b, c, and e), and Kruskal-Wallis test with Dunn correction (for multiple comparisons between the control group and other groups) (d).

[0231] Figure 3 This study demonstrates how PtdIns4P regulates the stability of ATGL proteins in the Golgi apparatus. (a) Quantification of PtdIns4P levels by immunostaining in HEK293-AAV cell types after glucose-free medium treatment (150 cells in each group). (b) PI4KB and SACM1L(c) Quantification of ATGL in HEK293T cell types 72 hours after knockdown (by immunoblotting) (control n=8, other groups n=4). (d) Quantification of ATGL in HEK293T cell types 24 hours after treatment with 5 µM PIK93, 10 µM BF738735, and 10 µM UCB9608 (by immunoblotting) (n=6 for all groups). (e) Quantification of ATGL in HEK293-AAV cell types by immunofluorescence 24 hours after UCB9608 treatment (61 cells used in both groups). (f) Quantification of ATGL in WCE, Golgi fraction, and vesicle fraction extracted from HEK293T cell types 6 hours after UCB9608 treatment (by immunoblotting) (n=6 for both groups). PI4KB and SACM1L 72 hours after knockdown, ATGL in HepG2 cell types was quantified (by Western blotting) (control n=8, other groups n=4). (g) 24 hours after treatment with 5 µM PIK93, 10 µM BF738735, and 10 µM UCB9608, ATGL in HepG2 cell types was quantified (by Western blotting) (for all groups n=6). (h) Pi4kb 72 hours after knockdown, ATGL in iBAs was quantified (by immunoblotting) (control group n=8, other groups n=4). (i) Pi4kb NEFA levels released by basal iBAs in starved medium 72 hours after knockdown (for all groups n=6). (j) Sacm1l 72 hours after knockdown, ATGL in iBAs was quantified (by immunoblotting) (control group n=6, other groups n=4). (k) Sacm1l NEFA levels released by basal iBAs in starved medium 72 hours after knockdown (n=6 for all groups). (l) ATGL quantification in iBAs (by immunoblotting) 24 hours after treatment with 1 µM PIK93, 1 µM BF738735, and 1 µM UCB9608 (n=6 for all groups). (m) NEFA levels released by basal iBAs in starved medium 24 hours after treatment with 1 µM PIK93, 1 µM BF738735, and 1 µM UCB9608 (n=6 for all groups). Data are presented as mean ± sem and analyzed using paired t-tests (e), Mann-Whitney tests (a and d), ANOVA with Dunnett correction (for multiple comparisons between control and other groups) (b, c, f–k and m), and Kruskal-Wallis tests with Dunn correction (for multiple comparisons between control and other groups) (l).

[0232] Figure 4 : Shows the Golgi resident E3 ligase complex CUL7 FBXW8 Polyubiquitinated ATGL is used for degradation. (a) Quantification of ATGL protein levels in HEK293T WCE by small-scale siRNA screening using Western blotting (control n=18, other groups n=3). (b) CUL7 and FBXW8 Immunofluorescence micrographs of ATGL cells in the HEK293-AAV cell type 72 hours after knockdown (61 cells in each group). Scale bar, 5 µm. (c) CUL7 and FBXW8 Immunoblots of ATGL in WCE, Golgi apparatus, and vesicle fractions extracted from HEK293T cells 72 hours after knockdown (n=6 in both groups). An asterisk (*) indicates a nonspecific band. (d) CUL7 and FBXW8 Immunoblot analysis of ATGL in HepG2 cell types 72 hours after knockdown (control group n=8, other groups n=4). (e) Cul7 and Fbxw8 72 hours after knockdown, immunoblotting of ATGL in iBAs (control group n=8, other groups n=4). (f) Cul7 and Fbxw8 NEFA levels released by basal iBAs in starved medium 72 hours after knockdown (for all groups n=6). (g) Glucose-free medium, CUL7 absent. FBXW8 Six hours post-treatment, ATGL immunoblotting was performed on HEK293T cell types (n=5 for all groups). Data are presented as mean ± sem and analyzed using two-tailed unpaired t-tests (b and g), paired t-tests (c), ANOVA with Dunnett correction (for multiple comparisons between control and other groups) (a, d, and e), and Kruskal-Wallis test with Dunn correction (for multiple comparisons between control and other groups) (f).

[0233] Figure 5(a–b) Quantification of CUL7 and FBXW8 in WCE, Golgi fraction, and vesicle fractions extracted from HEK293T cell type after treatment with glucose-free medium (a) or UCB9608 (b) (n=6 for all groups). Asterisk * indicates nonspecific bands. (c) Western blot analysis of PIP bands incubated with recombinant protein FBXW8-FLAG and FBXW8 mutant-FLAG. (d–e) Quantification of FBXW8-FLAG (d) and FBXW8 mutant-FLAG (e) in WCE, Golgi fraction, and vesicle fractions extracted from HEK293T cell type after treatment with glucose-free medium (n=6 for both groups). (f) A schematic diagram illustrating the working model of the mechanism by which glucose availability is coupled to lipolysis via Golgi PtdIns4P. Results are shown as mean ± sem and analyzed using paired t-tests (a, b, d, and e).

[0234] Figure 6 This demonstrates the manipulation of the Golgi apparatus PtdIns4P-CUL7. FBXW8 -ATGL axis regulates ATGL-driven lipolysis and fatty liver development. (a) Two weeks after AAV8 administration, hepatocyte-specific Pi4kb Quantification of ATGL protein in the liver of knockout mice (by immunoblotting) (n=8 in both groups). (b) Hepatocyte-specific... 2 weeks after AAV8 administration Cul7 and Fbxw8 Quantification of ATGL protein in the liver of knockout mice (by immunoblotting) (n=6 for all groups). (c) ATGL protein in hepatocytes 1 hour after 2-DG injection. Cul7 and Fbxw8 Quantification of ATGL protein in the liver under double knockout background (by immunoblotting) (n=6 for all groups). (d) Determination of liver TG levels in NCD-fed mice 4 weeks after AAV8 administration or HFD-fed mice 8 weeks after AAV8 administration (n=10 for both NCD groups and n=5 for both HFD groups). (e) Hepatocyte-specific effects of AAV8 administration on NCD and HFD-fed mice. Cul7 and Fbxw8Measurement of liver TG levels in knockout mice (n=11 for all groups in NCD, n=5 for all groups in HFD). (f) Schematic diagram of the experimental design for administering UCB9608 to wild-type mice. (g) Plasma FFA levels in wild-type mice 8 weeks after administration of DMSO and UCB9608 (n=12 for both groups). (h) Liver TG levels in mice treated with DMSO and UCB9608 (n=15 for both groups). Data are presented as mean ± sem and analyzed using two-tailed unpaired t-tests (a, c, d, and h), Mann-Whitney test (g), and ANOVA with Dunnett correction (for multiple comparisons between the control group and other groups) (b and e).

[0235] Figure 7 This study illustrates the amelioration of NASH progression and steatosis in rats with human liver grafts by blocking the production of PtdIns4P in the Golgi apparatus. (a) Schematic diagram of the experimental design for administering UCB9608 to wild-type mice after inducing NASH via HFD, high glucose / fructose water, and thermoneutral feeding. (b) Weight gain during UCB9608 treatment (n=10 in both groups). (c) EcoMRI analysis of lean body mass, fat body mass, and body weight in NASH mice (n=10 in both groups). (d) Plasma FFA levels 12 weeks after UCB9608 administration (n=10 in both groups). (e) Plasma TG levels 12 weeks after UCB9608 administration (n=10 in both groups). (f) Plasma ALT and AST activities in NASH mice after UCB9608 administration (n=10 in both groups). (g) Liver weight / body weight ratio in NASH mice after UCB9608 administration (n=10 in both groups). (h) Determination of TG levels in the livers of NASH mice after UCB9608 administration (n=10 in both groups). (i) Histological scores of livers of NASH mice treated with DMSO and UCB9608 (n=10 in both groups). (j) Transcriptional levels of inflammatory markers in NASH livers determined by qPCR (n=10 in both groups). (k) Transcriptional levels of fibrosis markers in NASH livers determined by qPCR (n=10 in both groups). (l) Schematic diagram of the experimental design for ex vivo administration of UCB9608 to human liver grafts during normothermic machine perfusion. (m–n) Quantification of ATGL protein (by immunoblotting) and TG levels in human liver biopsies obtained at different time points in two lobes. Results are shown as mean ± sem and analyzed using two-tailed unpaired t-tests (b to g, j (Ccl2 and Tnfα) and k (Col1a2, Timp1 and Acta2)), Mann-Whitney test (h, i, j (Emr1 and Il1b) and l (Tgfb1 and Col1a1)) and Pearson's correlation (n) analysis.

[0236] Figure 8 The study demonstrates how Golgi PtdIns4P regulates the stability of ATGL protein in the Golgi apparatus. (a) Quantification of PtdIns4P levels in HEK293-AAV cell types by immunostaining after 24 hours of treatment with 5 µM PIK93, 10 µM BF738735, and 10 µM UCB9608 (136 cells used in all groups). (b) Quantification of Golgi region in HEK293-AAV cells by anti-GM130 immunostaining after 72 hours of treatment with 5 µM PIK93, 10 µM BF738735, and 10 µM UCB9608 (100 cells used in all groups). (c) Quantification of ectopically expressed ATGL in HEK293T cell types (by immunoblotting) after 24 hours of treatment with 5 µM PIK93, 10 µM BF738735, and 10 µM UCB9608 (n=3 for all groups). Results are presented as mean ± sem and analyzed using ANOVA with Dunnett correction (for multiple comparisons between the control group and other groups) (c) and Kruskal-Wallis test with Dunn correction (for multiple comparisons between the control group and other groups) (a and b).

[0237] Figure 9 : This demonstrates that glucose deprivation increases ATGL protein in the Golgi apparatus via PtdIns4P regulation. (a) Quantification of ectopically expressed ATGL in HepG2 cell types (by immunoblotting) after treatment with 5 µM MPIK93, 10 µM BF738735, and 10 µM UCB9608 for 24 hours (n=3 for all groups). Results are shown as mean ± sem and analyzed using ANOVA with Dunnett correction (for multiple comparisons between control and other groups) (a). Example

[0238] Example 1: Glucose deprivation increases ATGL-driven lipolysis levels

[0239] Previous work has demonstrated that intracellular fatty acids (FAs) regulate ATGL-driven lipolysis via peroxisome FA oxidation, thereby linking FA availability with FA release. To elucidate whether other general cell-autonomous mechanisms regulate lipolysis in response to the availability of other nutrients such as glucose, mice were fasted for 24 hours, and plasma free fatty acid (FFA) levels, an alternative marker of lipolysis, were quantified. The inventors observed a significant upregulation of FFA levels during fasting. Figure 1a). Next, the levels of lipolytic proteins in the liver and adipose tissue of fasted mice were analyzed, including ATGL, phosphorylated HSL, MGL, perilipin 1 (PLIN1), and comparative gene identification 58 (CGI-58). Interestingly, only ATGL showed significantly increased protein levels in interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT), and liver in response to prolonged fasting. Figure 1 (b) This indicates that the general sensory system regulates ATGL-driven lipolysis to meet cellular energy demands. Both iWAT and gonadal white adipose tissue (gWAT) showed slightly upregulated pHSL levels, indicating some level of altered hormone signaling. To understand the timeline of HSL phosphorylation and ATGL upregulation in response to prolonged glucose deprivation, mice were treated with 2-deoxy-D-glucose (2-DG), a non-hydrolyzable glucose analog that effectively blocks glucose utilization and thus mimics glucose deprivation in vivo. Lipolysis and lipolytic protein levels were analyzed at different time points following 2-DG treatment. Similar to fasting, glucose deprivation significantly and rapidly induced lipolysis after 2-DG treatment. Figure 1 c) indicates that the molecular pathways involved may not be dependent on transcriptional regulation. To analyze the hormone axis, the inventors first determined plasma insulin levels and observed a decrease in insulin levels only 1 hour after 2-DG administration, suggesting that only insulin was involved. Therefore, the inventors next quantified the major players involved in lipolysis in the liver and adipose reservoir at different time points. Interestingly, the upregulation of HSL phosphorylation was observed only 1 hour after 2-DG administration in white adipose tissue, while ATGL protein levels increased in the liver and iBAT 1 hour later and showed further accumulation in the liver, iBAT, iWAT, and gWAT in response to prolonged glucose deprivation. Figure 1 (d) This indicates that HSL only partially participates in this altered lipolysis response as an immediate modulator, while ATGL regulation maintains lipolysis at high levels during glucose deprivation through a mechanism still unknown. Therefore, the immediate effects of insulin on mice with prolonged glucose deprivation 6 hours after 2-DG treatment were tested. Unexpectedly, lipolysis levels were only partially inhibited, and 30 minutes after insulin treatment, the levels of pHSL and ATGL remained unchanged against the background of glucose deprivation. Figure 1 e). Furthermore, 6 hours after co-injection of 2-DG and insulin, under prolonged glucose deprivation conditions, the anti-lipolytic effect of insulin was completely absent. Figure 1These observations led the inventors to hypothesize the existence of an intrinsic cellular mechanism regulating ATGL-driven lipolysis, independent of hormone signaling, which is effective to varying degrees in both short-term and long-term glucose deprivation-induced lipolysis. More importantly, the inventors obtained subcutaneous fat from patients before and after normoglycemic hyperinsulinic clamp (EHC), corresponding to low and high circulating glucose states, respectively. Interestingly, higher ATGL levels were observed in subcutaneous fat tissue obtained before EHC in patients with either insulin sensitivity or insulin resistance. Figure 1 (g, h), indicating that similar ATGL regulation occurred in humans as observed in mouse models.

[0240] To gain a better understanding of the mechanisms underlying ATGL regulation in these organs, the inventors examined the liver and adipose tissue of fasted or glucose-deficient mice. Atgl Transcript levels, and found Atgl Transcript levels remained unchanged in adipose tissue, further indicating that regulation is independent of transcriptional events. Conversely, prolonged fasting and glucose deprivation promoted changes in liver levels. Atgl Transcript levels, which may be due to the activation of hepatic PPARα in response to FA signaling transduction in the liver. Since adipose tissue is known to contribute to FA release into plasma, the inventors examined whether upregulation of ATGL in adipose tissue, rather than the liver, promotes an increase in plasma FFA levels during glucose deprivation. Therefore, they utilized liver and adipose tissue specificity... Atgl Knockout mice (respectively) AtglLKO and AtglAKO In mice, it was found that only adipose tissue ATGL was required to increase plasma FFA levels in response to glucose deprivation. In summary, the existence of a general intrinsic cellular mechanism for regulating ATGL-driven lipolysis independently of insulin signaling by detecting glucose availability was demonstrated.

[0241] Example 2: Glucose deprivation increases the level of ATGL protein in the Golgi apparatus.

[0242] Since ATGL protein levels are primarily regulated independently of transcription in glucose-deprived mice, the inventors focused on the mechanisms of ATGL stabilization in response to glucose availability. Given the complexity of mouse models, the inventors first examined whether glucose deprivation-induced ATGL stabilization could be reproduced in the HepG2 cell type (an immortalized human hepatocyte cell line). Consistent with in vivo data, removal of glucose from the culture medium and 2-DG treatment were observed to strongly induce ATGL protein abundance without altering transcript levels, suggesting that post-translational regulation of ATGL is similar to that in liver and adipose tissue. Figure 2a, b). This process was further confirmed in immortalized brown adipocytes (iBAs), where increased lipolysis levels and ATGL protein abundance were observed, although this was observed after glucose utilization was blocked. Atgl Transcription levels remained unchanged. Figure 2 c, d, e, f). Furthermore, in response to glucose deprivation, the regulation of the ATGL protein is also replicated in both HEK293T and HEK293-AAV cell types (two immortalized human embryonic kidney cell types). Figure 2 In addition, the inventors deprived the culture medium of serum, amino acids, and fatty acids and observed that serum deprivation alone promoted the accumulation of intracellular ATGL levels, suggesting that glucose and serum deficiency induce the release of FA from TG reserves through enhanced ATGL-driven lipolysis via the same mechanism.

[0243] Following synthesis in the ER, ATGL is transported to the Golgi apparatus and then translocates to the surface of the LD within the Golgi apparatus. Inhibition of this process strongly reduces intracellular ATGL protein levels, highlighting the importance of the Golgi-LD axis in regulating ATGL abundance and function. Therefore, the inventors next focused on the LD and the Golgi apparatus as potential sites where ATGL protein levels may be regulated during glucose deprivation. First, the inventors isolated LD from the HepG2 cell type and observed an increase in ATGL levels in the LD fraction during glucose deprivation, suggesting that ATGL may be regulated on the LD. Therefore, the inventors treated the HepG2 cell type with DGAT1 and DGAT2 inhibitors, key enzymes in FA esterification to produce TG, to block TG production, thereby depleting LD abundance within 48 hours. Interestingly, even in the absence of LD, glucose deprivation still increased endogenous ATGL. Conversely, HepG2 cells treated with BFA (brefidobacterium A) (an antibiotic that blocks Golgi apparatus formation) did not upregulate intracellular ATGL levels upon glucose deprivation, indicating that ATGL abundance is regulated at the Golgi apparatus level rather than on the LD surface. Therefore, the inventors quantified ATGL levels in the Golgi apparatus using immunostaining and fractionation, and observed significantly elevated protein levels in both the Golgi apparatus and vesicle regions in response to glucose deprivation. Figure 2 (i, j). These data demonstrate that ATGL is primarily regulated at the Golgi level, and then translocates to functional sites on vesicles and their LD surfaces to regulate lipolysis.

[0244] Example 3: Decreased PtdIns4P levels in the Golgi apparatus promote ATGL stability

[0245] It has been reported that both glucose and serum deprivation reduce Golgi PtdIns4P levels in various cell types, primarily mediated by promoting SACM1L retention in the Golgi apparatus after transfer from the ER. Based on this knowledge and our observation that both glucose and serum deprivation induce ATGL upregulation, we hypothesize that the reduction in Golgi PtdIns4P levels during nutrient deprivation may stabilize ATGL and, in turn, promote lipolysis. First, the inventors examined Golgi PtdIns4P levels using a P4M-EGFP sensor and a PtdIns4P antibody in the HEK293-AAV cell type. Consistent with previous publications, a decrease in Golgi PtdIns4P levels was observed after glucose removal from the culture medium, as indicated by live-cell imaging and immunostaining, and the enrichment of SACM1L in the Golgi apparatus. Figure 3 a). These observations are supported by a decrease in the total amount of PtdIns4P following glucose deprivation. Glucose deprivation reduces Golgi PtdIns4P levels, independent of the AMPK pathway, as activation and inhibition of this kinase do not affect PtdIns4P levels in the Golgi apparatus. To investigate the role of Golgi PtdIns4P in lipolysis, the inventors genetically manipulated Golgi PtdIns4P levels through siRNA-mediated knockdown of PI4KB (the main lipid kinase produced by Golgi PtdIns4P) and SACM1L. Interestingly, a decrease in Golgi PtdIns4P was observed to lead to an increase in ATGL levels, while accumulation of Golgi PtdIns4P decreased the abundance of ATGL protein in the HEK293T cell type. Figure 3 b). Furthermore, the inventors used three PI4KB inhibitors to block the production of PtdIns4P in the Golgi apparatus, and all three compounds increased the levels of both endogenous and ectopic ATGL protein. Figure 3 c and Figure 8 a, b, c). Similar to glucose deprivation, when the synthesis of PtdIns4P in the Golgi apparatus is blocked, ATGL protein accumulates in the Golgi apparatus ( Figure 3 (d, e) This indicates that Golgi PtdIns4P levels are crucial for determining ATGL stability in the Golgi apparatus. Since glucose deprivation promotes ATGL-mediated lipolysis in different cell types, the effect of Golgi PtdIns4P levels on this process in hepatic and adipocyte types was examined. It was confirmed that a decrease in Golgi PtdIns4P increased both endogenous and ectopic ATGL levels, while upregulation of the former had the opposite effect on ATGL stability in hepatocyte types. Figure 3 f, g and Figure 9 a). Similarly, genetic depletion and pharmacological inhibition of PI4KB promoted ATGL stability and lipolysis levels, while SACM1L depletion inhibited this process in adipocytes ( Figure 3 h–m).

[0246] Considering the relationship between glucose deprivation and Golgi PtdIns4P, and their roles in ATGL stability, the inventors next investigated whether glucose deprivation could regulate ATGL degradation via Golgi PtdIns4P. They first analyzed ATGL regulation in response to glucose deprivation in the presence of SACM1LK2A overexpression, a Golgi-resident mutant of SACM1L used to reduce Golgi PtdIns4P levels. Furthermore, they examined the ATGL response to glucose deprivation by depleting Golgi PtdIns4P through inhibition of UCB9608-mediated PtdIns4P formation. The loss of ATGL regulation following SACM1LK2A overexpression and UCB9608 treatment together suggest that a decrease in Golgi PtdIns4P levels is not only necessary for glucose deprivation-induced ATGL upregulation, but also sufficient to increase ATGL levels even under high glucose conditions. In summary, it can be seen that glucose availability regulates the distribution of SACM1L between the ER and the Golgi apparatus, thereby regulating the level of PtdIns4P in the Golgi apparatus, which in turn controls lipolysis by regulating the stability of ATGL in the Golgi apparatus.

[0247] Example 4: E3 ligase complex CUL7 FBXW8 Regulating ATGL in the Golgi apparatus

[0248] Although ATGL is regulated in the Golgi apparatus, how Golgi PtdIns4P levels regulate ATGL stability remains unclear. Considering that proteasome-mediated degradation and autophagy are major pathways that can influence intracellular protein degradation, the inventors blocked both processes by treatment with MG132 and chloroquine, respectively. Interestingly, only MG132 treatment resulted in cellular accumulation of ATGL protein, indicating that ATGL degradation is proteasome-mediated. Post-translational modification of substrates via K48-linked polyubiquitination by E3 ligase serves as a major signal for proteasome-mediated degradation. Therefore, the inventors examined whether lysine ubiquitination of ATGL is irreducible for its regulation under glucose deprivation. Thus, the inventors constructed a lysine-insensitive ATGL mutant and analyzed its accumulation after treatment with glucose-free medium or in response to the addition of 2-DG. In contrast to wild-type ATGL, the lysine-insensitive ATGL mutant was unresponsive to either treatment, indicating that lysine modification is essential. Furthermore, K48-linked polyubiquitination of ATGL was reduced under glucose deprivation. These observations suggest the presence of a Golgi-resident E3 ligase that senses glucose availability and can polyubiquitinate ATGL for proteasome-mediated degradation.

[0249] To identify the E3 ligase responsible for glucose deprivation-induced ATGL accumulation in the Golgi apparatus, the inventors conducted a small screening of E3 ligases reportedly localized to the Golgi apparatus and known Golgi PtdIns4P effectors. Among the candidates tested, only CUL7 and FBXW8 (E3 ligase complex CUL7) were observed. FBXW8 The depletion of the components significantly increased the ATGL levels in whole-cell extracts and Golgi fractions. Figure 4 (a–c). Notably, while ATGL levels were elevated in both the Golgi apparatus and the vesicular moiety, CUL7 and FBXW8 were barely detectable in the vesicular moiety. Furthermore, loss of either component significantly reduced LD levels, depending on the presence of ATGL. The scaffold protein CUL7 interacts with the E3 ligase RBX1, the adaptor protein SKP1, and the substrate receptor FBXW8 to form a functional E3 ligase complex, the formation of which is crucial for stabilizing these proteins. Additionally, a positively charged multi-functional region at the N-terminus of FBXW8, i.e., the putative interactor of the negatively charged PtdIns4P, allows CUL7 to... FBXW8 The E3 ligase complex senses glucose availability by being recruited to the Golgi apparatus. This makes the complex a promising candidate for regulating glucose deprivation-induced ATGL-driven enhancement of lipolysis. Before testing this hypothesis, the inventors first analyzed CUL7. FBXW8 Functions in liver and fat cell types. CUL7 FBXW8 The depletion of these proteins led to higher ATGL protein levels and a decrease in ATGL-dependent abundance of LD and lipolysis. Figure 4 (d–f). These findings led the inventors to further explore CUL7. FBXW8 To understand the mechanistic role of ATGL regulation, we measured FBXW8 (CUL7) via co-immunoprecipitation (Co-IP). FBXW8 The inventors observed a physical interaction between the substrate acceptor of the E3 ligase complex and ATGL. FBXW8 Following depletion, the direct interaction between FBXW8 and ATGL, and the reduced polyubiquitination level of the K48-linked ATGL, demonstrate that CUL7 FBXW8 It acts as an E3 ligase complex and interacts with ATGL in the Golgi apparatus for polyubiquitination and proteasome-mediated degradation. Finally, the inventors examined the effect in the absence of CUL7. FBXW8 ATGL regulation of glucose deprivation response was found to eliminate glucose sensing under these conditions. Figure 4 g). In summary, these data show that the E3 ligase complex CUL7 FBXW8It is essential for regulating polyubiquitination and degradation of ATGL in the Golgi apparatus in response to glucose deprivation.

[0250] Example 5: Golgi PtdIns4P regulation of CUL7 FBXW8 Function

[0251] CUL7 and FBXW8 have previously been shown to be localized in the Golgi apparatus of certain cell types. To understand CUL7... FBXW8 To investigate how glucose availability is sensed and subsequently ATGL degradation, the inventors next examined the cellular localization of CUL7 and FBXW8 using immunostaining. Consistent with previous reports and our grading results, endogenous CUL7 was highly enriched in the Golgi apparatus rather than in vesicles. Figure 4 c). However, unexpectedly, both endogenous and ectopic expressed FBXW8 showed a dispersed pattern, with partial localization in the Golgi apparatus ( Figure 4 c). Conversely, the vesicle portion contains almost no detectable levels of endogenous FBXW8 protein (c). Figure 4 c). Considering the presence of a multi-region at the N-terminus of FBXW8, the inventors hypothesize that the Golgi apparatus PtdIns4P may be able to recruit FBXW8 to the Golgi apparatus, where it will be assembled into a functional E3 ligase complex with CUL7, thereby enabling CUL7 to... FBXW8 It is capable of sensing glucose availability and regulating ATGL levels. Therefore, the inventors examined the levels of CUL7 and FBXW8 in the Golgi apparatus after glucose deprivation by fractionation and immunostaining. As expected, FBXW8, but not CUL7, showed a decreased level in the Golgi apparatus. Figure 5 a). Next, the inventors depleted the Golgi apparatus PtdIns4P and observed a decrease in the distribution of FBXW8 into the Golgi apparatus ( Figure 5 (b) indicates that its distribution depends on Golgi PtdIns4P. To demonstrate the interaction between the multivariate region of FBXW8 and PtdIns4P, the inventors constructed FLAG-labeled wild-type FBXW8 and FBXW8 mutants, in which all lysine and arginine residues in the multivariate region of FBXW8 were mutated to alanine, and these were stably expressed in HEK293T cell type. The inventors purified recombinant FBXW8 protein from stable cell lines and examined their binding ability to various lipid species in membrane lipid bands. Among these tested lipid species, PtdIns4P showed binding ability to wild-type FBXW8, while the interaction between PtdIns4P and mutant FBXW8 was low. These data were confirmed using PIP bands, which also demonstrated that PtdIns4P binding ability to mutants was reduced compared to wild-type FBXW8 ( Figure 5c). In addition, the inventors used PtdIns4P conjugated agarose beads to precipitate mutant and wild-type FBXW8, and the inventors showed that PtdIns4P beads also exhibited a stronger interaction with wild-type FBXW8 compared to mutants.

[0252] After demonstrating the physical interaction between the multi-region of FBXW8 and PtdIns4P, the inventors next examined whether the recruitment of FBXW8 to the Golgi apparatus was affected by glucose availability. Consistent with the interaction ability of PtdIns4P, wild-type FBXW8 levels in the Golgi apparatus decreased upon glucose deprivation, while mutant FBXW8 was insensitive to glucose levels. Figure 5 d, e). In summary, glucose deprivation leads to lower levels of PtdIns4P in the Golgi apparatus. This negatively charged phospholipid interacts with the positively charged multi-regional domains of FBXW8 and influences the recruitment of FBXW8 to the Golgi apparatus, where CUL7 FBXW8 The E3 ligase complex is assembled into polyubiquitinated ATGL for proteasome degradation. Figure 5 f).

[0253] Example 6: Golgi apparatus PtdIns4P and CUL7 FBXW8 Regulation of ATGL-driven lipolysis in mice

[0254] Data identified Golgi PtdIns4P as a central player in regulating ATGL-driven lipolysis, thus coupling glucose availability and intracellular FA release to regulate energy homeostasis. Therefore, the inventors next focused on hepatic ATGL regulation as a key factor in hepatic lipolysis and TG accumulation, particularly under the conditions of fatty liver pathogenesis. To control Golgi PtdIns4P levels in the liver, the inventors administered AAV8-U6-gRNA-TBG-Cre virus. LSL-spCAS9 In mice, the gRNA library specifically targets hepatocytes Pi4kb or Sacm1l This method resulted in effective depletion of PI4KB protein levels in the liver two weeks after AAV8 administration. Figure 6 a). In the absence of PI4KB, ATGL protein levels were significantly upregulated ( Figure 6 a). Reported Sacm1l Overall depletion of SACM1L led to embryonic lethality, and liver-specific depletion of SACM1L also resulted in death two weeks after AAV8 administration, hindering any mechanistic studies and demonstrating the association of SACM1L with other pathways. Therefore, the inventors then used the same strategy described above to specifically knock out SACM1L in hepatocytes. Cul7 and Fbxw8 The depletion of CUL7 leads to instability of FBXW8 in the liver, but vice versa. Figure 6b). This contradicts previous reports that demonstrated these two factors could stabilize each other. The inventors observed an increase in ATGL levels after knocking out Cul7 and Fbxw8 in the liver. Figure 6 b), indicating CUL7 FBXW8 Their effects in vivo are consistent. Therefore, we next examine the E3 ligase complex CUL7. FBXW8 Whether hepatic ATGL upregulation is responsible for responding to glucose deprivation. Therefore, we administered 2-DG to wild-type and Cul7 Double knockout of Fbxw8 mice for 1 hour. This treatment resulted in upregulation of ATGL levels in the liver of wild-type mice, but CUL7 was absent. FBXW8 Then no ( Figure 6 c) Emphasizing CUL7 FBXW8 Its fundamental role in regulating glucose deprivation-induced ATGL stability.

[0255] It has been reported that upregulation of liver ATGL levels is highly effective in reducing TG deposition in fatty liver. Therefore, the inventors [did something related to] PtdIns4P-CUL7 in the Golgi apparatus. FBXW8 Following genetic and pharmacological manipulation of the -ATGL axis, the physiological and pathophysiological effects were examined in a non-alcoholic fatty liver disease (NAFLD) model. The inventors induced hepatocyte-specific... Pi4kb Quantitative analysis of liver TG levels after PI4KB knockout. In fact, PI4KB depletion reduces liver TG levels ( Figure 6 d). Furthermore, when PI4KB was ablated in a mouse model of hepatic steatosis induced by a 12-week high-fat diet (HFD) model, TG deposition in the fatty liver was significantly reduced 8 weeks after viral administration. Figure 6 d). Similarly, the inventors measured under NCD rearing conditions. Cul7 and Fbxw8 Hepatic TG levels in knockout mice were measured, and it was found that only CUL7 ablation reduced hepatic TG levels. Figure 6 e). Conversely, depletion of either factor reduced liver TG accumulation in HFD-treated mice for 12 weeks ( Figure 6 e). In addition to gene manipulation, the inventors also examined the effects of PI4KB inhibitors on lipolysis regulation and NAFLD progression. Therefore, wild-type mice were fed HFD for 12 weeks to induce hepatic steatosis and then treated with UCB9608 (a PI4KB inhibitor reportedly effective in mouse models with organ transplantation). Figure 6 f). Interestingly, PI4KB inhibition significantly increased plasma FFA levels (f). Figure 6The presence of g), indicating that Golgi PtdIns4P plays a consistent in vivo role in regulating ATGL-driven lipolysis in adipose tissue. Therefore, the inventors compared ATGL protein levels in different adipose deposits and livers from mice treated with DMSO and UCB9608, and found that the latter showed significantly higher ATGL protein levels in iBAT, gWAT, and liver, with a similar trend in iWAT. Consistent with reported upregulation of ATGL in adipose tissue, UCB9608 treatment resulted in higher energy expenditure, likely due to increased FA levels for energy burning. Enhanced ATGL-driven lipolysis in adipose tissue may contribute to increased FA release and excretion, while elevated hepatic ATGL levels may drive intrahepatic lipolysis and reduce hepatic TG levels. Therefore, the inventors measured hepatic TG levels in the livers of UCB9608-treated mice and observed substantially reduced hepatic steatosis, which requires the presence of ATGL in the liver ( Figure 6 h). In summary, this proves that the Golgi apparatus PtdIns4P-CUL7 FBXW8 - The ATGL axis regulates hepatic ATGL levels and LD clearance, which may provide a potential target for improving hepatic steatosis.

[0256] Example 7: Reduction of PtdIns4P in the Golgi apparatus weakens NASH progression

[0257] Having demonstrated the potential applicability of the identified NAFLD treatment pathway, the inventors hope to further investigate whether targeting the production of PtdIns4P in the Golgi apparatus can attenuate the progression of NASH from simple hepatic steatosis. NASH progression is characterized by increased inflammation and fibrosis, and currently lacks approved pharmacological treatments (Loomba, R). et al Cell184, (2021)). To mimic NASH in mouse liver, wild-type mice were fed a heat-neutralized HFD diet combined with drinking water containing high glucose and fructose for 24 weeks, followed by treatment with UCB9608 for an additional 12 weeks. Figure 7 a). Interestingly, a significant reduction in body weight and fat mass was observed in patients treated with UCB9608, consistent with the enhanced lipolysis and energy expenditure induced by UCB9608. Figure 7 (b–d) suggests the effective role of UCB9608 in metabolic improvement. Therefore, the inventors further examined plasma TG levels and ALT and AST activities in NASH mice. The reduction in these parameters after UCB9608 treatment indicated reduced hepatic steatosis and damage, leading the inventors to perform histopathological analysis of livers from treated NASH mice, comparing them with livers from control NASH mice treated with the vector (b–d). Figure 7e, f). The inventors first measured liver weight, liver TG, and fatty acid oxidation levels before histological analysis, and observed that UCB9608 treatment reduced the liver weight / body weight ratio and liver TG levels, while significantly increasing fatty acid oxidation (e, f). Figure 7 g, h). Next, the inventors performed histological analysis, including hematoxylin and eosin (H&E) staining, Oil Red O staining (ORO), cleaved caspase 3, and Gomori green trichrome staining, to determine the grade of hepatic steatosis, inflammation and injury, and fibrosis. Interestingly, the livers of mice treated with UCB9608 showed significantly lower grades of steatosis, liver injury, inflammation, fibrosis, and NAFLD activity, indicating that UCB9608 treatment inhibited NASH progression, although the NASH induction in these mouse livers was relatively low compared to clinical NASH biopsies from human patients. Figure 7 i). The reduction in inflammation levels after UCB9608 treatment was further confirmed by quantifying the transcript levels of inflammatory markers. Figure 7 Similarly, liver fibrosis, a crucial pathophysiological index defining NASH, is also analyzed by quantifying the transcriptional levels of fibrosis markers. Consistent with this, UCB9608 treatment significantly improved the expression levels of fibrosis-related markers in the livers of treated NASH mice compared to vector-treated control NASH mice. Figure 7 k).

[0258] Progression of NASH can lead to cirrhosis and potentially hepatocellular carcinoma, both of which require liver transplantation to avoid liver failure. However, liver grafts cannot be transplanted when the liver graft is fatty liver, thus requiring potential strategies to reduce fatty liver for clinical use. Given the ability of UCB9608 to improve hepatic TG levels in a mouse model, the inventors developed a perfusion system (Clavien, PA) et al Nat Biotechnol 40, (2022) tested the lipid-reducing effect of UCB9608 on human fatty liver in an in vitro process. The inventors obtained discarded human liver grafts with 15% macrocystic steatosis and perfused the liver with blood for 8 days. UCB9608 was administered daily to achieve a target concentration of 10 µM ( Figure 7The grafts showed normal bile acid excretion and oxygen consumption, indicating graft health during perfusion. Interestingly, the inventors detected non-esterified fatty acids (NEFAs) in the dialysate during perfusion, suggesting that induced lipolysis led to FA release. To investigate the time course of hepatic lipid reduction, the inventors performed biopsies from both lobe sections to determine ATGL and TG levels. Consistent with the above findings in mice, the inventors observed a significant increase in ATGL levels and a decrease in TG deposition following UCB9608 treatment, while ATGL and hepatic TG levels remained unchanged in the two control grafts, indicating that ATGL-driven lipolysis mediated lipid reduction following UCB9608 administration. Figure 7 In summary, the discovery of Golgi PtdIns4P-mediated ATGL stabilization in response to glucose deprivation enhances novel therapeutic strategies to combat NASH progression by blocking Golgi PtdIns4P production and also serves as a potential therapeutic approach for ex vivo repair of fatty liver grafts.

[0259] in conclusion

[0260] It is well known that the endocrine system plays a crucial role in glucose and fatty acid (FA) processing during various nutritional states. Elevated plasma glucose triggers insulin secretion from β-cell types to promote glucose uptake in multiple tissues, including adipose tissue and the liver. Insulin also promotes de novo lipogenesis to utilize excess glucose as a carbon source for FA production, but simultaneously inhibits lipolysis to reduce the release of FA from lipid reserves. Conversely, glucagon secreted from α-cell types under hypoglycemic conditions, or norepinephrine released from the sympathetic nervous system in response to starvation or cold exposure, increases lipolysis via phosphorylation of ATGL and HSL in hepatocytes and adipocytes. Typically, these extracellular hormones rapidly elicit a systemic response to acute physiological changes. However, hormone signaling typically does not function long-term due to desensitization of the corresponding receptors. Examples of this process include classical β-repressor-mediated desensitization of β1 and β2 adrenergic receptors and non-classical desensitization of β3 adrenergic receptors through significant transcriptional downregulation of receptor gene expression upon ligand binding. This may explain the inventors' results that elevated pHSL levels in adipose tissue were reversed during prolonged glucose deprivation. Such a homeostatic feedback loop may require cells to develop intrinsic mechanisms to modulate lipolysis to nutrient availability, as a complementary process to extracellular hormone-mediated regulation of lipid metabolism, enabling organisms to better meet complex physiological needs. In fact, the inventors have identified a novel intrinsic mechanism involving the central role of Golgi PtdIns4P, which senses glucose levels and ligates to the E3-linked enzyme complex CUL7. FBXW8Interactions. This intrinsic mechanism may further shape our understanding of the precise contribution of insulin signaling to the regulation of lipolysis, as insulin can also regulate intracellular glucose levels to initiate Golgi PtdIns4P-CUL7. FBXW8 The ATGL axis is used for lipolysis regulation. Decreased HSL phosphorylation is considered a major mediator of insulin's anti-lipolytic effect. Here, the inventors found that insulin does not inhibit pHSL under glucose deprivation conditions, suggesting that glucose itself is an important mediator of lipolysis and may mediate insulin's anti-lipolytic effect. This insulin signaling-independent mechanism may be particularly relevant to insulin resistance, as we found elevated ATGL levels in patients with type 2 diabetes during fasting. However, a key mediator of insulin signaling, insulin receptor substrate 1 (IRS1), is reported to be CUL7. FBXW8 The substrates of the study indicate crosstalk between the regulation of insulin and Golgi PtdIns4P-mediated lipolysis.

[0261] As a key mediator of ATGL-driven lipolysis regulated by Golgi PtdIns4P, FBXW8 is recruited to the Golgi apparatus to assemble the E3 ligase complex CUL7. FBXW8 Previous reports have indicated that CUL7 FBXW8 Assembly facilitated the stability of CUL7 and FBXW8, as the loss of either factor strongly reduced the protein level of the other. In fact, the inventors found that CUL7 depletion led to FBXW8 instability in all cell types used in this work, including the liver. As a result, FBXW8 levels were reduced in the Golgi apparatus and WCE because FBXW8 is poorly stable in the cytosol and did not assemble into the CUL7 scaffold. Conversely, the inventors found that FBXW8 depletion may only destabilize CUL7 in iBAs, highlighting the more significant role of the scaffold protein CUL7 in stabilizing the E3 ligase complex. Functionally, CUL7 also had a strong effect on ATGL-driven lipolysis, as CUL7 depletion in vitro and in the liver in HepG2 and HEK293AAV cell types resulted in lower LD abundance or TG levels compared to FBXW8 ablation.

[0262] Adipose tissue stores a large amount of fatty acids (FAs) in lipoproteins (LDs) and primarily supplies intracellular and extracellular FAs through lipolysis to meet the energy needs of its own cells and other tissues. Compared to adipocytes, most non-adipocytes have less LD, therefore, under glucose or serum deprivation, they must initiate autophagic degradation of non-essential cellular components to provide a carbon source for the production of mitochondrial-near LDs, allowing any intracellular lipolysis to facilitate the oxidation of FAs near fuel fatty acids. The inventors discovered that ATGLs are stabilized in the Golgi apparatus of starved cells. Once LDs are produced, this response can trigger lipolytic activation in starved cells, allowing ATGLs to translocate from the Golgi apparatus to LDs. These mechanisms ensure that less essential cellular components, in addition to FAs taken from circulation, are mobilized promptly and effectively to overcome nutrient deprivation in non-adipocytes. Furthermore, abundant LDs can be artificially induced in non-adipocytes using an oleic acid-containing medium. Under these conditions, glucose or serum starvation induces autophagic degradation of the LD coat proteins perilipin proteins 2 and 3 (PLIN2 / 3), the clearance of which clears the LD surface for ATGL localization and action. However, the inventors observed that PLIN1, the major LD shell protein in adipocytes, did not show differences in protein levels from various fat stores after fasting, indicating that the regulation of lipolysis in adipocytes and non-adipocytes exhibits unique characteristics despite the existence of common pathways. Furthermore, lipophage can directly mobilize fatty acids (FAs) from LDs via LAL1. In hepatocytes, lipolysis is responsible for releasing FAs from large LDs, and then FAs from the resulting smaller LDs are subsequently mobilized via lipophage.

[0263] NASH typically progresses from simple hepatic steatosis and is characterized by elevated hepatic lipids, inflammation, and fibrosis. Osteogenesis imperfecta is primarily due to elevated fatty acid delivery from the circulation, along with increased de novo lipogenesis in hepatocytes, decreased hepatic lipolysis, and decreased fatty acid oxidation. Inflammation is induced by the secretion of inflammatory factors and activation of inflammasomes in the Kupffer cell type, while the hepatic stellate cell type is responsible for collagen secretion, which leads to fibrosis, believed to be exacerbated by inflammation. Based on the inventors' findings and other reported studies, Golgi PtdIns4P may serve as an effective target for blocking multiple factors contributing to NASH progression. The inventors demonstrate that reducing Golgi PtdIns4P stabilizes ATGL and promotes intrahepatic lipolysis and LD clearance. Golgi PtdIns4P also plays a crucial role in NLRP3 activation through direct recruitment to the Golgi apparatus; therefore, inflammation in NASH livers may be directly alleviated by reducing PtdIns4P levels. Furthermore, procollagen is synthesized in the ER and transported to the Golgi apparatus, where it is processed and secreted into the extracellular space via large transport vesicles. The formation of these vesicles may require the action of Golgi PtdIns4P to induce membrane curvature, as in certain types of Golgi-derived vesicles. If so, targeting PI4KB is a very promising strategy to directly attenuate steatosis, inflammation, and fibrosis in NASH, as observed by the inventors in their mouse model. More importantly, UCB9608 treatment reduced plasma TG levels in the mouse NASH model, unlike the elevated TG secretion and hypertriglyceridemia observed after administration of ACC inhibitors in mice and humans.

[0264] Furthermore, the inventors' research indicates that UCB9608 can reduce fatty liver during ex vivo perfusion. This could allow for the use of more grafts for transplantation after testing more grafts perfused with UCB9608, as current fatty liver conditions are unsuitable for such clinical applications. Interestingly, UCB9608 is also known to have immunosuppressive properties. Considering that immunosuppressants are essential for preventing allogeneic graft rejection after organ transplantation, continued administration of UCB9608 post-transplantation would be a promising option if it is approved for clinical use.

[0265] In summary, the inventors' discovery identifies a novel intrinsic pathway that links glucose availability to lipid availability via glucose sensing through the Golgi PtdIns4P-ATGL-lipolysis pathway, which leads to the upregulation of lipolysis and increased release of fatty acids (FAs) from lipid reserves upon glucose depletion. In this manner, cells can process nutrient fluxes better than relying solely on paracrine hormone-mediated regulation of nutrient availability.

[0266] Materials and methods

[0267] EHC study

[0268] This study was approved by the local ethics committee (Bratislava, Slovakia) and complied with the ethical guidelines of the Declaration of Helsinki 2000. All study participants witnessed written informed consent before entering the study. Insulin sensitivity (M value) was determined using EHC. Briefly, human insulin (Actrapid 100 IU / mL, Bagswede NovoNordisk, Denmark) was infused into the antecubital vein in a pretreated continuous manner at a dose of 1 mU / kg / min. Blood glucose was measured in samples from the contralateral antecubital vein at 5-minute intervals and maintained at normal blood glucose (5.0 ± 0.25 mM) using a variable infusion rate of 20% glucose. Systemic insulin sensitivity (M value) was calculated from the steady-state plasma glucose infusion rate required to maintain normal blood glucose and normalized to steady-state insulinemia, expressed as per kg body weight per minute. Subcutaneous AT samples were collected from the abdominal region by needle aspiration biopsy during basal fasting (before insulin and glucose administration) and during the steady-state of EHC. Immediately rinse the samples, blot them, freeze them in liquid nitrogen, and store them at -80°C. Based on the insulin sensitivity index (M value), twenty-four middle-aged sedentary men (age 35.5 ± 1.5 years, BMI 28.9 ± 1.1 kg·m²) were included. -2 The obese population (25.7 ± 1.6%) was divided into insulin-sensitive (n = 16, M value 0.14 ± 0.02 mg / kg BW / min / insulin µU / ml) and insulin-resistant (n = 8, M value 0.04 ± 0.01 mg / kg BW / min / insulin µU / ml) subgroups.

[0269] Human liver perfusion

[0270] Human liver grafts discarded from various Swiss transplant centers were perfused for 8 days. The use of discarded grafts was approved by the local authority (Ethics Committee of the Canton of Zurich, KEK Nr. 2017-000412). The grafts had an initial weight of 2740 g (with gallbladder) before perfusion and were diagnosed by the Department of Pathology at the University Hospital of Zurich with 15% large cystic steatosis. The liver was obtained after a total ischemic time of 40 minutes following donor circulatory death. It was flushed with IGL-1 solution and perfused with Belzer MPS at 8°C for 2 hours. The inferior vena cava was closed with sutures, and the hepatic artery, portal vein, superior vena cava, and common bile duct were cannulated with steel cannulas. Perfusion was then performed with whole blood and a custom-made room temperature perfusion machine (Eshmuminov, D). et al(Nat Biotechnol 38, (2020)) Perfusion graft. Hepatic artery pressure was controlled at 80 / 50 mmHg, allowing a mean flow rate of 0.25 L / min. The vasodilator epoprostol was automatically infused to maintain stable arterial blood flow. Oxygenation was automatically controlled using an oxygen setpoint partial pressure equal to 12 kPa. Portal vein flow was controlled at 1 L / min, with a mean pressure of 2 mmHg. In addition, portal vein blood oxygenation was automatically regulated to achieve 65% saturation in the vena cava. Pressure regulation in the vena cava was further allowed using an electrically operated clamp valve to maintain a constant pressure of 0 mmHg. Glucose levels were automatically controlled to maintain 7 mmol / L from the second day of perfusion using a real-time glucose sensor (CCIT) and automated insulin infusion. Hematocrit was controlled by removing waste metabolites using a dialysis bypass and by using a fixed dialysate inflow rate of 200 mL / h and automatically controlled effluent. Liver function was maintained well for at least 6 days, and cell viability was maintained until the end of perfusion, as monitored by ATP analysis and histology. Eight days after perfusion, the liver weight decreased to 1507 g (without gallbladder) and was sent for pathological examination. The liver graft ultimately showed less than 5% of the large cystic steatosis as determined by the Department of Pathology at the University Hospital of Zurich.

[0271] Human liver perfusion—drug and perfusion fluid composition

[0272] The perfusion solution was mixed with 6 units of erythrocyte concentrate (A+), 5 units of fresh frozen blood cells, 1 unit of platelets, and 200 mL of 20% human albumin (CSL Behring). Following priming, an initial bolus of 2.25 g tazobactam and 500 mg methylprednisolone was administered. During perfusion, 1000 U heparin was perfused hourly. Additionally, 2.25 g tazobactam, 3.36 g ursodeoxycholic acid, and 500 mg methylprednisolone were continuously infused daily. UCB9608 was administered as a daily bolus by adding 300 µL of a 50 mM solution directly to the blood reservoir to achieve a final concentration of 10 µM in the perfusion solution. A larger bolus of 600 µL was administered on the first day.

[0273] Mouse experiment

[0274] All animal experiments were conducted with permission from the Veterinary Office of the State of Zurich. Both male and female mice were used in the experiments, and they were housed under standard conditions (22°C, 12-hour reverse light / dark cycle, dark period beginning at 7 am), on a normal diet (18% protein, 4.5% fiber, 4.5% fat, 6.3% ash, Provimi Kliba SA) and with water, in ventilated cages, 2–5 littermates per cage. The health of all mouse strains was monitored regularly according to FELASA guidelines. 8–10-week-old C57BL / 6N male mice were either fasted for 24 hours or injected with 2-DG (2 g / kg BW) to induce glucose deprivation for varying durations. AAV8-U6- Pi4kb gRNA-TBG-Cre, AAV8-U6- Sacm1l gRNA-TBG-Cre, AAV8-U6- Cul7 gRNA-TBG-Cre and AAV8-U6- Fbxw8 gRNA-TBG-Cre viral reservoir at 3×10 11 Genome copy / mouse dose intravenously injected female ROSA26-LSL-spCas9 Mice (Jackson Lab strain) expressed six gRNAs targeting a single gene and CAS9 in the liver. Two weeks later, livers were harvested after a 6-hour fast for protein and RNA extraction. To determine liver TG levels under NCD feeding conditions, livers were harvested 4 weeks after AAV administration, after a 6-hour fast. Also 4 weeks after AAV administration, before harvesting livers for protein extraction, liver-specific Cul7 and Fbxw8 double knockout mice were injected with 2-DG (2 g / kg BW) to induce 1 hour of glucose deprivation.

[0275] Male mice were used for NAFLD induction by feeding them HFD. AAV-U6- Cul7 gRNA-TBG-Cre and AAV-U6- Fbxw8 gRNA-TBG-Cre viral reservoir at 3×10 11 Genome copy / mouse dose injection ROSA26-LSL-spCas9 Mice were fed in HFD for 12 weeks, and their livers were harvested after a 6-hour fast. To investigate PI4KB in NAFLD progression, male mice were subjected to hepatocyte-specific depletion of PI4KB before intravenous administration of AAV8 to induce it. ROSA26-LSL-spCas9Mice were fed HFD for 12 weeks to induce fatty liver. After an additional 8 weeks, the mice were dissected after a 6-hour fast to harvest the liver. Similarly, male C57BL / 6N wild-type mice and liver-specific Atgl knockout mice (AtglLKO) were challenged with HFD for 12 weeks to induce fatty liver, followed by administration of UCB9608 mixed with HFD (5 mg / kg) for 8 weeks. The mice were then dissected after a 6-hour fast to collect tissue and blood samples.

[0276] Will Atgl floxed mice (Jackson Lab strain) were crossed with albumin-Cre (Jackson Lab strain) and Adip-CreERT2 strains to obtain, respectively Alb-Atgl fl / fl and AdipcreERT2-Atgl fl / fl mice (Sitnick, MT) et al Diabetes 62, (2013)), which depletes ATGL protein in the liver and fat stores (induced after tamoxifen tube feeding). Male mice from two strains were injected with 2-DG (2 g / kg BW) to induce glucose deprivation for 6 hours prior to tissue and blood collection.

[0277] 2-DG and insulin administration

[0278] Wild-type C57BL / 6N mice were treated with saline or 2-DG (2 g / kg BW) via intraperitoneal injection for 5.5 hours, followed by insulin treatment (Actrapid, human insulin, dose 0.75 U / kg BW) for 30 minutes. Mice were then euthanized to harvest gonadal white adipose tissue and blood to investigate the acute anti-lipolytic effect of insulin in the context of glucose deprivation. To investigate the long-term anti-lipolytic effect of insulin during glucose deprivation, wild-type C57BL / 6N mice were co-treated with 2-DG (2 g / kg BW) and insulin (0.75 U / kg BW) for 6 hours. Mice were then euthanized for blood sampling.

[0279] Application of UCB9608 in a mouse NASH model

[0280] To mimic NASH in rat liver, male C57BL / 6N mice were challenged for 24 weeks with HFD in drinking water containing 18.9 g / L glucose and 23.1 g / L fructose under thermoneutral feeding conditions (29°C), followed by administration of UCB9608 (15 mg / kg) via HFD for 12 weeks. Body composition measurements of the mice were then analyzed on EcoMRI, followed by fasting for 6 hours, and subsequent dissection to harvest liver and blood samples.

[0281] Indirect calorimetry

[0282] Before indirect calorimetry measurements were performed using the Phenomaster (TSE system) according to the manufacturer's instructions, 8–10-week-old male C57BL / 6N mice were fed HFD mixed with DMSO or UCB9608 (5 mg / kg) for 3 weeks. Animals were individually caged and acclimatized to their cages for 48 hours prior to metabolic recording. Energy expenditure between groups was analyzed using ANCOVA, and relative energy expenditure (RER) was analyzed using two-way ANOVA.

[0283] Cell culture

[0284] The immortalized mouse brown preadipocytes (iBAs) were kindly provided by Professor Klein's laboratory (Klein, J.). et al (BioEssays 24, (2002)). iBAs were cultured on collagen-coated plates in DMEM medium (high glucose) containing 10% FBS and 1% Pen / Strep. Once preadipocytes reached confluence, adipogenesis was induced for 2 days in differentiation medium containing IBMX (500 µM), dexamethasone (1 µM), insulin (20 nM), T3 (1 nM), and indomethacin (125 µM). iBAs were then refreshed with maintenance medium containing insulin (20 nM) and T3 (1 nM). On day 5, differentiated iBAs were re-seeded to reduce cell density. All experiments were routinely performed on day 9. For siRNA-mediated knockdown experiments, siRNA (100 nM) was delivered mediated by Lipofectamine RNAiMAX reagent to knock out the target gene in iBAs for 72 hours after recovery from re-coating for one day. To deprive iBAs of glucose, we added 2-DG to the culture medium at a specified concentration, or refreshed the cells with DMEM medium containing different glucose concentrations prepared from DMEM medium (Gibco, A1443001) with the same concentration of insulin and T3 as the maintenance medium.

[0285] HEK293T and HEK293-AAV cell types were cultured in DMEM (high glucose) with 10% FBS and 1% Pen / Strep. HEK293-AAV cell types were used for imaging, while HEK293T cells were used for the remaining work. To overexpress EGFP-P4M, EGFP-SACM1L, EGFP-SACM1LK2A, ATGL-FLAG, ATGL-HA, ATGLK0-HA, and FBXW8-FLAG in HEK cells, these plasmids were delivered at 60–80% cell confluence using Lipofectamine 2000 for 24 hours according to the manufacturer's instructions, followed by specific treatments based on the experimental design. To image the abundance of LD, HEK293-AAV cell types were induced to produce LD for 24 hours prior to 4% PFA fixation by 200 µM oleic acid (OA, O1008, from Sigma) conjugated with bovine serum albumin (BSA, A6003, from Sigma). Cells were then stained with BODIPY 493 / 503 and Hoechst 33342 for LD and nuclear labeling, respectively. Under other conditions, HEK cells were not treated with OA-containing medium. To deprive HEK cells of glucose, 2-DG was added to the medium at a specified concentration, or cells were refreshed with DMEM medium containing 4.5 g / L and 0 g / L glucose, prepared using the same DMEM medium (Gibco, A1443001). To deprive amino acids or fatty acids, we used amino acid-free medium (FUJIFILM, 048-33575) and added 5% fatty acid-free BSA (A6003, Sigma) to normal medium, respectively.

[0286] HepG2 cell types were cultured on collagen-coated plates in RPMI medium containing 10% FBS and 1% Pen / Strep. Lipofectamine RNAiMAX reagent-mediated siRNA (100 nM) was delivered to the cells in suspension for 48 hours. Unlike HEK cells, LD was not induced by OA treatment because LD is present in HepG2 cell types in normal medium. The inventors treated HepG2 cells for 48 hours with 10 µM PF04620110 (DGAT1 inhibitor, Sigma) and PF-06424439 (DGAT2 inhibitor, Sigma) or for 6 hours with 5 µg / mL BFA (B5936, Sigma) to deplete LD and intact Golgi apparatus in HepG2 cell types, respectively, before glucose deprivation. To deprive HepG2 cells of glucose, we added 2-DG to the culture medium at a specified concentration, or refreshed the cells with RPMI medium containing different glucose concentrations prepared from the same RPMI medium (Gibco, 11879020).

[0287] All cell lines used in this work were tested negative for mycoplasma contamination on a regular basis. All siRNAs used in these cell lines are listed in Table 1.

[0288] Table 1: siRNA sequences used in this study.

[0289] RNA extraction and quantitative PCR

[0290] Total RNA was extracted from fat banks, liver, or in vitro cell cultures using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Genomic DNA was removed from crude RNA samples by DNase treatment (NEB BioLabs). For a single sample, 1 µg of total RNA was converted into a cDNA library using a high-capacity cDNA reverse transcription kit (Applied Biosystems, 4368813). Quantitative PCR was performed using the KAPA SYBR FAST Universal Kit (Roche, KK4618) on a ViiA7 (Applied Biosystems), and relative transcription levels were normalized to 36B4 expression using the ΔΔCt method. Primer sequences are listed in Table 2.

[0291] Table 2: qPCR primer sequences used in this study.

[0292] Molecular cloning

[0293] Human ATGL-FLAG and ATGLK0-FLAG expression constructs were generated as previously described (Ding, L). et al (NatMetab 1-14 (2021)). Based on these vectors, the inventors designed primers to replace the FLAG tag with an HA tag and inserted them into the pEGFP-N1-FLAG vector (Addgene, #60360) with a stop codon before the FLAG-EGFP CDS to obtain the ATGL-HA and ATGLK0-HA expression constructs. Human SACM1L and SACM1LK2A CDS were cloned from the vector mCherry-FKBP-SAC1 (Addgene, #108127) and inserted into the pEGFP-C1-FLAG vector (Addgene, #46956) to have an EGFP tag at the N-terminus. Human FBXW8-FLAG CDS was cloned from the HEK293T cDNA library and inserted into the pLenti-CMV-MCS-BSD vector to obtain the pLenti-FBXW8-FLAG construct. The human FBXW8 mutant CDS, with N-terminal lysine and arginine replaced by alanine, was purchased from Vector Builder Inc. The CDS was then inserted into the pLenti-CMV-MCS-BSD vector to obtain the pLenti-FBXW8 mutant-FLAG construct. Scarlet-macroprotein CDS was cloned from plasmid pmScarlet-macroprotein-C1 (Addgene #85048) and inserted into the pLenti-CMV-MCS-BSD vector to obtain the pLenti-Scarlet-macroprotein construct. Six different gRNA sequences targeting single genes were ordered from Microsynth AG and transcribed, then inserted into the AAV-U6-TBG-Cre vector to construct AAV-U6-Pi4kb gRNA-TBG-Cre, AAV-U6-Sacm1l gRNA-TBG-Cre, AAV-U6-Cul7 gRNA-TBG-Cre, and AAV-U6-Fbxw8 gRNA-TBG-Cre. All plasmids used for cell culture were extracted using the NucleoBond Xtra-Midi kit (Macherey-Nagel, 740410.100).

[0294] Viral packaging and stable cell line construction

[0295] For lentiviral packaging, the inventors used a third-generation system. pLenti-Scarlet-macroprotein, pLenti-FBXW8-FLAG, or pLenti-FBXW8 mutant-FLAG plasmids were transfected into HEK293T cell lines in OptiMEM medium via PEI with pMD2.G (Addgene, #12259) and psPAX2 (Addgene, #12260). Two days later, the virus-containing medium was collected and concentrated in PEG-it virus precipitation solution (SBI, LV825A-1) according to the manufacturer's instructions. The concentrated lentivirus was added to HEK cell culture and promoted with polybrene (Sigma, H9268). Two days later, the cells were resuspended and selected with 10 µg / mL blastomycin (ThermoFisher, A1113903).

[0296] For AAV packaging, the inventors used the HEK293-AAV cell type (Cell Biolabs). AAV8 serotype helper plasmids were purchased from Plasmid Factory. AAV-U6-Pi4kb gRNA-TBG-Cre, AAV-U6-Sacm1l gRNA-TBG-Cre, AAV-U6-Cul7 gRNA-TBG-Cre, AAV-U6-Fbxw8 gRNA-TBG-Cre, or AAV-U6-NS gRNA-TBG-Cre plasmids were transfected into the HEK293-AAV cell line in OptiMEM medium via PEI along with the helper plasmids. The medium was collected after 4–5 days and concentrated using the AAVaced concentration reagent (SBI, AAV110A-1) according to the manufacturer's instructions. The titer of the concentrated virus was determined by quantitative PCR using the Addgene titer assay protocol.

[0297] Golgi apparatus and LD extraction

[0298] According to the manufacturer's instructions, the Golgi apparatus and Golgi-associated vesicles were isolated using a small Golgi enrichment kit (GO-037, Invent Biotechnologies). Approximately 3 × 10⁻⁶ 7 HEK293T cell types were used for fractionation. Microspheres containing Golgi bodies and vesicles were directly dissolved in RIPA buffer and sonicated for 10 minutes before denaturation in Lämmeli buffer. The target protein was analyzed by Western blotting as described below.

[0299] LD separation was performed as previously described (Bersuker, K). et alDevelopmental Cell 44, (2018)). In short, HepG2 cells were harvested in cold PBS and centrifuged at 500×g for 10 min to pellet the cells. The cells were then incubated in cold hypotonic lysis medium (HLM, 20 mM Tris-HCl pH 7.4 and 1 mM EDTA) with a complete protease inhibitor (Roche). After homogenization in multiple rotations, the homogenate was centrifuged at 1000×g for 10 min, and the supernatant was collected for further ultracentrifugation via HLM at different sucrose concentrations. The floating fraction was collected and finally resuspended in 1% SDS containing RIPA buffer for Western blot analysis.

[0300] Immunoblotting, immunoprecipitation, co-immunoprecipitation, and immunofluorescence

[0301] Fat stores, liver fragments, differentiated iBAs, HepG2, HEK293T, and HEK293-AAV cell types were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1.0% Triton X100, 0.5% sodium deoxycholate) with a protease inhibitor (Complete, Roche) and a phosphatase inhibitor (Halt phosphatase inhibitor mixture, ThermoFisher). The homogenate was centrifuged at 12,000 × g for 10 min at 4°C to remove debris and whole-cell extract (WCE) was collected. The concentration of WCE was determined by DC protein analysis (Bio-Rad). After boiling the samples in Lämmeli buffer at 95°C for 5 min, an equal volume of protein was loaded and separated on 12% SDS-PAGE.Proteins were transferred onto nitrocellulose membranes and analyzed using ATGL (1:1000, Cell Signaling), CGI-58 (1:1000, Proteintech), phosphorylated HSL (Ser660) (1:1000, Cell Signaling), PLIN1 (1:1000, Cell Signaling), HSL (1:1000, Cell Signaling), γ-tubulin (1:10000, Sigma), FLAG (1:10000, Sigma; 1:1000, Cell Signaling), MGL (1:1000, Abcam), HA (1:1000, Cell Signaling), FBXW8 (1:100, Santa Cruz; 1:1000, Sigma), CUL7 (1:100, Santa Cruz), and HSP90 (1:1000, Cell Signaling). Signaling), K48 polyubiquitin (1:1000, Cellsignaling), PI4KB (1:1000, Sigma), SACM1L (1:1000, Sigma), AKT1 (1:1000, CellSignaling), phosphorylated AKT1 (Ser473) (1:1000, CellSignaling), TGN46 (1:1000, Abcam), MAOA (1:1000, Abcam), PLIN2 (1:1000, Abcam), PMP70 (1:10000, Sigma), cadherin (1:1000, CellSignaling), ARF1 (1:1000, Invitrogen), GAPDH (1:1000, CellSignaling), RAB7 (1:1000, CellSignaling). Western blots were performed using α1-sodium potassium ATPase (1:1000, Abcam), cleaved caspase 3 (1:1000, CellSignaling), phosphorylated AMPKα (Thr172) (1:1000, Cell Signaling), AMPKα (1:1000, CellSignaling), and GM130 (1:1000, Proteintech). Raw antibody signals were observed using HRP-conjugated secondary antibody (1:5000, Millipore) and an ImageQuant system (GE Healthcare Life Sciences).

[0302] To immunoprecipitate FLAG-labeled ATGL or FBXW8, HEK293 cells were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1.0% Triton X100) with a protease inhibitor (Complete, Roche) and a phosphatase inhibitor (Halt phosphatase inhibitor mixture, ThermoFisher). The homogenate was centrifuged at 12,000 × g for 10 min at 4°C to remove debris and WCE was collected. The concentration of WCE was determined by DC protein analysis (Bio-Rad). After washing three times with cold RIPA buffer, 20 µl of anti-FLAG beads (A2220, Sigma) were added to the WCE and incubated overnight at 4°C by rotation. The beads were then washed six times with cold RIPA buffer. Proteins were eluted by boiling the beads at 95°C for 5 min with 2 × Lämmeli buffer. Then, immunoblotting was performed using K48-specific ubiquitin antibody (1:1000, Cell Signaling), γ-tubulin (1:10000, Sigma), ATGL (1:1000, Cell Signaling), FBXW8 (1:1000, Sigma), and FLAG (1:1000, Cell Signaling) to analyze the eluted proteins.

[0303] To perform immunostaining of ATGL, GM130, CUL7, FBXW8, and FBXW8-FLAG in HEK293-AAV cell types or stable cell lines expressing Scarlet-macroprotein after various treatments, HEK293-AAV cell types were fixed with 4% PFA for 20 min and permeabilized with 0.25% PBST (Triton X100 in PBS) or 20 µM digitalisin (for ATGL immunostaining only) for 20 min. After washing three times with cold PBS, cells were blocked with 1% BSA for 1 h and incubated overnight at 4°C with ATGL antibody (1:100, Cell Signaling), GM130 (1:100, Proteintech), CUL7 antibody (1:50, Santa Cruz), FBXW8 antibody (1:100, Sigma), and FLAG antibody (1:100, Cell Signaling). After washing three times with cold PBS, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (1:200, ThermoFisher) at room temperature for 1 hour. Finally, after washing three times with PBS to remove residual secondary antibody, cells were stained with Hoechst 33342 to label the nuclei. We measured the ATGL intensity / Golgi apparatus area ratio or the FBXW8 intensity / Golgi apparatus area ratio to quantify Golgi ATGL or FBXW8 levels, respectively, and the ratios of the experimental groups are shown as relative values ​​to the control. Similarly, we immunostained GM130 cells in the HepG2 cell type using the same method, except that we incubated them with Alexa Fluor 568 conjugated secondary antibody (1:200, ThermoFisher) for 1 hour.

[0304] To immunostain ATGL in iBAT, tissues were fixed overnight at 4°C with 4% PFA after dissection. Samples were then dehydrated with 10% and 30% sucrose solutions and cryosectioned to a thickness of 15 µm. Slides were washed once in PBS for 5 minutes, three times in 0.1% PBST, and blocked for 1 hour with 10% normal donkey serum (NDS) diluted in PBST. ATGL antibody (1:100, Cell Signaling) was then diluted in blocking solution and incubated overnight at 4°C. Residual antibody was washed three times with PBST 1 hour before application of Alexa Fluor 568 conjugated secondary antibody (1:200 in 5% NDS, ThermoFisher). Finally, slides were treated with mounting solution after three washes with PBST.

[0305] All photos were acquired using Olympus Fluoview 3000 confocal microscopy and processed using ImageJ.

[0306] PtdIns4P determination

[0307] HEK293-AAV cell type was co-expressed with Scarlet-macroprotein and EGFP-P4M, followed by glucose deprivation. Cell live imaging was performed, and Golgi PtdIns4P levels in individual cells were measured as the Golgi GFP intensity / cytoplasmic GFP intensity ratio. Golgi PtdIns4P levels after glucose deprivation are shown as relative values ​​to the ratio obtained from cells cultured in normal glucose medium.

[0308] To immunostain PtdIns4P cells under different conditions, HEK293-AAV cell line (Scarlet-macroprotein) was fixed with 4% PFA for 15 min, washed three times with PBS containing 50 mM NH4Cl, and then permeabilized with 20 µM digitalis saponin in PBS for 5 min. After washing three times with PBS, the cells were blocked with 5% normal goat serum (NGS) in PBS containing 50 mM NH4Cl for 1 h at room temperature. The primary antibody against PtdIns4P (1:100 in 5% NGS; Echelon Biosciences) was applied to the cells and incubated overnight at 4°C. After three washes, the cells were replaced with Alexa Fluor 488 conjugated secondary antibody (1:200 in 5% NGS; Thermo Fisher). Finally, the cells were washed three times and stained with Hoechst 33342 to label the nuclei. We determined the PtdIns4P intensity / Golgi area ratio to quantify the PtdIns4P level in the Golgi apparatus, and the ratio of the experimental group is shown as a relative value relative to the control.

[0309] The total mass of PtdIns4P from HEK293T cell line was determined using the PtdIns4P Mass ELISA kit (K-4000E, Echelon Biosciences) according to the manufacturer's instructions. Prior to analysis, total acidic lipids were extracted by TCA precipitation, methanol:CHCl3:HCl (80:40:1) extraction, and phase separation. The organic phase was dried, and the residue was dissolved in PBS-T buffer and sonicated for 10 min. The lipid samples were then measured and normalized to the total protein mass.

[0310] Membrane lipid banding, PIP banding, and pull-down assays using PtdIns4P agarose beads.

[0311] Wild-type and mutant FBXW8 were purified from an established stable HEK293T cell line using a FLAG purification kit (CELLMM2, Sigma) according to the manufacturer's instructions. After elution with buffer containing 3×FLAG peptides, the eluent was filtered and concentrated using a 10 kDa cutoff concentrator (Pierce, 88513). The concentrations of the purified recombinant proteins were determined by protein assay (Bio-Rad) before aliquoting.

[0312] The binding between recombinant protein and lipids was determined using membrane lipid strips (P-6002, Echelon Biosciences) and PIP strips (P-6001, Echelon Biosciences). Briefly, both strip membranes were blocked for 1 hour with blocking buffer (PBS containing 0.1% Tween 20 and 3% fat-free BSA). 3 µg of recombinant protein was diluted in fresh blocking buffer and applied to the strip membranes for 1 hour. The strip membranes were then washed three times with strip washing buffer (PBS containing 0.1% Tween 20) for a total of 30 minutes. The strip membranes were incubated for 1 hour with FLAG antibody diluted in strip washing buffer (1:1000, Cell Signaling), followed by three washes and an additional 1 hour incubation with HRP-conjugated secondary antibody (1:10000, Cell Signaling) as part of a routine immunoblotting procedure. The strip membranes were developed and captured using an ImageQuant system (GE Healthcare Life Sciences).

[0313] Recombinant protein was purified using PtdIns4P binding beads (P-B004, Echelon Biosciences), PC (phosphatidylcholine) binding beads (P-B0 PC, Echelon Biosciences), and control beads (P-B000, Echelon Biosciences) according to the manufacturer's instructions. Briefly, 100 µL of the slurry was precipitated by low-speed centrifugation, and the precipitate was resuspended in 300 µL of wash-binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.25% Igepal). 10 µg of recombinant wild-type and mutant FBXW8 were added, and the mixture was incubated at room temperature for 3 hours. The beads were precipitated and washed 6 times with wash-binding buffer. Finally, the protein was eluted by boiling the beads in 2×Lämmeli buffer at 95°C for 5 minutes. The eluted protein was then analyzed by Western blotting using a FLAG antibody (1:1000, Cell Signaling).

[0314] Lipolysis measurement

[0315] For in vitro lipolysis measurements, iBAs collected on day 9 were washed with pre-warmed PBS and then starved for 2 hours in phenol red-free DMEM medium (low glucose) containing 1% fatty acid-free BSA. After collecting the medium, non-esterified fatty acid release was measured using a NEFA assay kit (Wako NEFA kit) via a Synergy Gen5 Platerador and normalized to total protein mass. Similarly, NEFA levels were determined using the same kit to measure dialysis fluid from ex vivo perfusion of liver grafts collected at different time points.

[0316] Plasma insulin and glucose measurement

[0317] For mouse insulin measurements, plasma was collected from wild-type mice at different time points following 2-DG administration. Insulin levels were determined using a 5 µl plasma sample via an ultrasensitive mouse insulin ELISA kit (Crystal Chem, Cat #90080) according to the manufacturer's protocol. Absorbance was measured at 450 / 630 nm using a Synergy Gen5 Platerador. Glucose levels were determined using a 2 µl plasma sample via a glucose assay kit (Sigma, MAK263) according to the manufacturer's protocol. Absorbance was read at 570 nm using a Synergy Gen5 Platerador.

[0318] Plasma levels of FFA, TG, ALT and AST activities

[0319] The levels of lipolysis and liver injury were determined using plasma collected from mice under different conditions. FFA levels were measured using an NEFA assay kit (Wako NEFA kit). TG levels were determined using a TG analysis kit (Roche Trig / GB reagent). ALT and AST activities were determined using ALT (MAK052, Sigma) and AST (MAK055, Sigma) assay kits according to the manufacturer's instructions. Absorbance was monitored using a Synergy Gen5 Platerador.

[0320] Determination of glucose, glucose-6-phosphate and glyceraldehyde-3-phosphate in the liver

[0321] Liver fragments (50–100 mg) were homogenized in 2 volumes of ice-cold PBS. The homogenized sample was centrifuged at 13,000 × g for 10 min to remove insoluble material. The supernatant was collected, and protein concentration was measured by a protein assay (Bio-Rad). The supernatant was then deproteinized using a 10 kDa MWCO spin filter (Pierce, #88513). Glucose levels were measured using 1 µl of the deproteinized sample via a glucose assay kit (Sigma, MAK263). Glucose-6-phosphate levels were measured using 10 µl of the sample via a glucose-6-phosphate assay kit (Sigma, MAK014) at 450 nm using a Synergy Gen5 Platereader, or via a glyceraldehyde-3-phosphate assay kit (Abcam, ab273344) using a Synergy Gen5 Platereader recording fluorescence in kinetic modes at 535 / 587 nm every 30 sec. The concentrations of glucose, glucose-6-phosphate, and glyceraldehyde-3-phosphate were normalized to the protein concentration.

[0322] Liver TG measurement

[0323] Before homogenizing each 50 mg of tissue with 1 mL of isopropanol, record the weight of liver fragments (50–100 mg). Incubate the homogenate by rotation at room temperature, then vortex at 2000 × g for 10 minutes to remove fragments and collect the supernatant. Measure TG levels in a SynergyGen5 Platerader (Roche trig / GB reagent) and normalize to the weight of the liver fragments.

[0324] Histological staining

[0325] Following tissue harvesting, livers from mice on a normal diet and NAFLD mice were fixed directly in 4% PFA in PBS for 24 hours. For hematoxylin and eosin (H&E) staining, fixed liver fragments were transferred to 65% ethanol, embedded in paraffin using a tissue processor and embedding machine, sectioned at 10 µm, and stained using standard protocols. For Oil Red O staining, the fixed livers were dehydrated sequentially with 10% sucrose and 30% sucrose solutions. Liver sections were cryogenically sectioned at 20 µm and stained with Oil Red O solution (O1516, Sigma).

[0326] For the NASH group, the liver was dissected and fixed with 4% PFA in PBS at 4°C for 24 hours. Samples were sent to the University of Zurich for histological analysis. In short, liver tissue was dehydrated and embedded in paraffin, and sectioned at 3 µm. Slides were stained directly with hematoxylin and eosin (H&E) and Gomori green trichrome, or stained with anti-cleavage caspase 3 after antigen retrieval. Images were then captured using a digital slide scanner and scored using the NASH Clinical Research Network Scoring System (Kleiner, DE). et al (Hepatology 41, (2005)) Quantifying the grades of fatty degeneration, ballooning degeneration, inflammation, and fibrosis. For ORO staining, fixed sample sections were stained with Oil Red O solution and then counterstained with eosin. Images were obtained using a digital slide scanner.

[0327] Quantitative and statistical analysis

[0328] For in vivo studies, littermates were randomly assigned to treatment groups in all experiments. Sample sizes were determined based on previous experiments, and the number of animals used in each experiment is indicated in the corresponding legend. For cell culture experiments, biological assays were performed at least three times in parallel (shown as n in the legend), and all cell culture experiments were performed in parallel using 2–3 times techniques for RNA and protein analysis. Unless otherwise specified, results are expressed as mean ± sem. When data were normally distributed, two-tailed unpaired or paired Student's t-tests were applied to comparisons between two groups; otherwise, the Mann-Whitney test or Wilcoxon test was used. When data were normally distributed, ANOVA was used for comparisons between multiple groups; otherwise, the Kruskal-Wallis test was used. All statistical analyses were performed using GraphPad Prism 7 software. Statistical differences are expressed as exact p-values ​​or no significance (ns).

[0329] References

[0330] Ding, L. et al . Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis. Nat Metab 1–14 (2021) doi:10.1038 / s42255-021-00489-2.

[0331] Loomba, R., Friedman, S. L. & Shulman, G. I. Mechanisms and diseaseconsequences of nonalcoholic fatty liver disease. Cell 184, 2537–2564 (2021).

[0332] Clavien, P.-A. et al . Transplantation of a human liver following 3days of ex situ normothermic preservation. Nat Biotechnol 40, 1610–1616(2022).

[0333] Eshmuminov, D. et al . An integrated perfusion machine preservesinjured human livers for 1 week. Nat Biotechnol 38, 189–198 (2020).

[0334] Sitnick, M. T. et al . Skeletal Muscle Triacylglycerol Hydrolysis DoesNot Influence Metabolic Complications of Obesity. Diabetes 62, 3350–3361(2013).

[0335] Klein, J., Fasshauer, M., Klein, H. H., Benito, M. & Kahn, C. R.Novel adipocyte lines from brown fat: A model system for the study ofdifferentiation, energy metabolism, and insulin action. BioEssays 24, 382–388(2002).

[0336] Renella et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology 78(6), 1966-1986 (2023).

[0337] All scientific publications and patent documents cited in this specification are incorporated herein by reference. In particular, EP2935269 B1, EP 2193131 B1 and Knight et al. 1006, Cell 125:733-747 are incorporated herein by reference.

[0338] sequence

[0339] In the event of any discrepancy between the sequences shown in this specification and the sequences according to the Closed Sequence Protocol of WIPO Standard ST.26, the sequences shown herein shall prevail.

Claims

1. A method for reducing fatty liver fat in vitro, the method comprising: - The fatty liver graft is perfused using a perfusion solution in the perfusion step, said perfusion solution comprising a compound selected from PI4KB inhibitors of formulas 1, 2, and 3, or a pharmaceutically acceptable salt or solvate thereof. (1)、 (2)、 (3)， in - Z can be (methoxy)(methyl)phenyl, (methyl)(trifluoromethoxy)phenyl, (methoxy)(methyl)pyridyl, (ethyl)(methoxy)pyridyl, (ethoxy)(methyl)pyridyl, or dimethoxypyridyl. - A 11 It can be H, methyl, ethyl, hydroxymethyl, or hydroxyethyl. - Q is a heterocyclic ring as shown in Equation 4. (4)， in X is N or C(H), Y is N or C(H), T is either S or N(CH3) - R 2 R 4 and R 5 Independently selected from H, methyl, and ethyl, - R 6a SO2R 8 , where R 6a In opposite or between positions - R 7a Selected from OH, OCH3, halogens, NH2, CH2-OH and NH-CO-R 8 , - Each R 7b Independently selected from OH, halogen, CN, NO2, C1-C6 alkyl, C1-C6 alkoxy, OCF3, OCH2CF3, OCH2Ph, OCH2-cyclopropyl, OCH2CH2OH, OCH2CH2NMe2, CF3, OCF3, C1-C6 alkyl-OH, NHSO2R 9 , NHCOR 8 and NR 9 R 10 , - c is 0 or 1 in R 8 C1-C6 alkyl or NR 9 R 10 ,and R 9 and R 10 Independently selected from H and C1-C6 alkyl groups.

2. The method according to claim 1, wherein the concentration of the PI4KB inhibitor in the perfusion fluid is from 0.1 to 100 µM.

3. The method of claim 3, wherein the initial concentration of the PI4KB inhibitor in the perfusion fluid is 0.1 to 100 µM.

4. The method of claim 2, wherein the concentration of the PI4KB inhibitor is controlled and adjusted during the perfusion step to a concentration of 1 to 40 µM in the perfusion solution.

5. The method according to any of the preceding claims, wherein the concentration of the PI4KB inhibitor is adjusted in the following manner - Add PI4KB inhibitor via bolus injection, wherein the PI4KB inhibitor is soluble in an organic solvent, particularly DMSO, or - Continuous application, wherein the PI4KB inhibitor is bound to albumin, or the PI4KB inhibitor is encapsulated in a nanoemulsion, or the PI4KB inhibitor is part of a nanoparticle.

6. The method according to any of the preceding claims, wherein the perfusion step is performed until a non-fatty liver is obtained, particularly for 2 to 14 days, more particularly for 8 days.

7. The method according to any of the preceding claims, wherein the perfusion fluid comprises a perfusion medium, wherein the perfusion medium is whole blood, leukocyte-anemic blood, leukocyte-free blood, or an oxygenated blood substitute.

8. The method according to claim 1, wherein the compound is selected from PI4KB inhibitors of formulas 1a, 2a, and 3. (1a)、 (2a) Among them, Z and A 11 Q, R 2 R 4 R 5 R 6a R 7a R 7b c is as defined above.

9. The method according to any of the preceding claims, wherein A 11 It can be methyl, ethyl, hydroxymethyl, or hydroxyethyl.

10. The method according to any of the preceding claims, wherein Z is selected from the portions of formulas Za, Zb, Zc, Zd, Ze, Zf, Zg, Zh, and Zi. (For)、 (Zb)、 (Zc)、 (Zd)、 (Yes)、 With(f)、 (Zg)、 (Zh)、 (Zi).

11. The method according to any of the preceding claims, wherein one of X and Y is N and the other is C(H).

12. The method according to any of the preceding claims, wherein Q is selected from heterocycles of formulas Qa, Qb, Qc, Qd, and Qe. (Qa)、 (Qb)、 (Qc)、 (Qd)、 (Yes).

13. The method according to any of the preceding claims, wherein R 6a SO2R 8 , where R 8 It is a C1-C6 alkyl group.

14. The method according to any of the preceding claims, wherein - R 7a Selected from OH, NH2 and OCH3, and / or - R 7b Selected from OH, halogen, NO2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl-OH, NH2, NHSO2R 9 and NHCOR 8 , where R 9 and R 8 As defined above.

15. The method according to any of the preceding claims, wherein the compound is selected from PI4KB inhibitors of formulas 1b, 2b, and 3. (1b)、 (2b)。

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