Use of deaminases and transaminases in treatment of polyglutamine diseases

Abstract

The present invention relates to a use of a class of deaminases or transaminases and cofactors or metabolic substrates thereof as drugs for treating polyglutamine (polyQ) diseases. Specifically, in the present invention, it is found that a series of transaminases and deaminases (asparagine synthetase (ASNS), etc.) can degrade polyQ aggregates by means of deamination of polyQ proteins, so as to treat polyQ diseases.

Description

Application of deaminases and transaminases in the treatment of polyglutamine diseases Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of a combination of deaminases or transaminases and their cofactors for the treatment of polyglutamine diseases. Background Technology

[0002] Neurodegenerative diseases seriously endanger the lives of millions of people worldwide. These complex diseases involve numerous genetic and non-genetic factors, but all share pathological protein aggregation or protein homeostasis problems (processes of protein synthesis, folding, breakdown, and degradation). One group of neurodegenerative diseases known as polyglutamine (PolyQ) diseases (including Huntington's disease) currently lacks viable treatments. Only adjunctive therapies addressing motor deficits can alleviate symptoms, failing to address the underlying neurological issues. Therefore, there is an urgent need to find new treatment options.

[0003] Therefore, there is an urgent need to develop new, safe, and effective drugs for the treatment of polyglutamine diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a class of safe and effective active ingredients for treating polyglutamine diseases.

[0005] A first aspect of the present invention provides the use of an active ingredient for preparing a formulation or composition, wherein the active ingredient is selected from:

[0006] (a) A transaminase and optionally a cofactor or metabolic substrate of the transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), protein-glutamine γ-glutamyltransferase 2 (TGM2); or combinations thereof;

[0007] (b) A deaminase and optionally a cofactor or metabolic substrate of the deaminase, wherein the deaminase is selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), a fusion gene (CAD) of carbamoyl-phosphate synthetase 2-aspartate transcarbamylase and dihydroorotase, human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpesvirus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), and Vibrio parahaemolyticus VopC. VopC), Burkholderia pseudomallei lethal factor 1 (BLF1), and Shigella flexneri OspI;

[0008] (c) A combination of components (a) and (b); and / or

[0009] (d) Aspartic acid;

[0010] And the formulation or composition is intended for use selected from the group consisting of:

[0011] (1) Convert some glutamine residues in polyQ protein aggregates into glutamate residues;

[0012] (2) Promotes the degradation of polyglutamine-containing proteins or their aggregates;

[0013] (3) Reduce the proportion of polyQ aggregate-positive cells in vitro;

[0014] (4) Reduce the neurotoxicity of polyglutamine protein or its aggregates; and / or

[0015] (5) Treatment of polyglutamine diseases.

[0016] In another preferred embodiment, the cofactor or metabolic substrate of the transaminase or deaminase includes:

[0017] Cofactors or metabolic substrates of asparagine synthase: aspartic acid, adenosine triphosphate (ATP);

[0018] Cofactor or metabolic substrate of phosphoaminotransferase: 5-phospho-alpha-D-ribose 1;

[0019] The cofactor or metabolic substrate of CTP synthase 1: uridine triphosphate (UTP);

[0020] The cofactor or metabolic substrate of CTP synthase 2: uridine triphosphate (UTP);

[0021] Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 1: D-fructose 6-phosphate;

[0022] Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 2: D-fructose 6-phosphate;

[0023] Cofactor or metabolic substrate of glutamine-dependent NAD+ synthase: deamido-NAD+;

[0024] Cofactor or metabolic substrate of GMP synthase: Xanthic acid (XMP);

[0025] The cofactor or metabolic substrate of the fusion gene (CAD) of carbamoyl synthase II-aspartate transcarbamoylase and dihydroorotase is hydrogen carbonate.

[0026] In another preferred embodiment, the polyQ protein aggregate is an insoluble polyQ protein aggregate.

[0027] In another preferred embodiment, the active ingredient is used to convert at least 1 / 30, preferably at least 1 / 10, more preferably at least 1 / 5, more preferably at least 1 / 4, more preferably at least 1 / 3, and most preferably at least 1 / 2 of the glutamine (Q) residues in the polyQ protein aggregate into glutamate (E) residues.

[0028] In another preferred embodiment, 1 / 30, preferably at least 1 / 10, of the glutamine (Q) residues are converted to glutamate (E) residues, thereby substantially degrading the insoluble polyQ protein aggregates.

[0029] In another preferred embodiment, the basic degradation refers to at least 60%, more preferably at least 70%, more preferably at least 80%, and more preferably at least 90% degradation of polyQ protein aggregates.

[0030] In another preferred embodiment, the proportion of polyQ aggregate positive cells is reduced by at least 1 / 3, preferably at least 1 / 2, after treatment with the active ingredient compared to untreated cells.

[0031] In another preferred embodiment, the formulation or composition is administered to a subject.

[0032] In another preferred embodiment, the subject's brain region has a higher level of polyglutamine protein or its aggregates.

[0033] In another preferred embodiment, the above-mentioned high content refers to a content significantly higher than the benchmark value.

[0034] In another preferred embodiment, the benchmark value is the corresponding content in normal subjects.

[0035] In another preferred embodiment, the content is significantly higher than the reference value of ≥6 / 5, preferably ≥5 / 4, more preferably ≥4 / 3, and even more preferably ≥3 / 2.

[0036] In another preferred embodiment, the subject is a mammal, more preferably a rodent (such as a mouse or rat) or a human.

[0037] In another preferred embodiment, the brain region includes the ventricles, cerebrospinal fluid, striatum, and other parts.

[0038] In another preferred embodiment, the polyglutamine-containing protein is selected from the group consisting of: Huntingtin (HTT) protein, TATA box-binding protein (TBP), atrophin-1, and androgen receptor (AR).

[0039] In another preferred embodiment, the polyglutamine disease is selected from the group consisting of: Huntington's disease (HD), six types of spinocerebellar ataxia (SCA) including types I, II, III, VI, VII and XVII; dentate nucleus-rubella-leucocerebellar Lewy body atrophy (DRPLA); and spinal bulbar muscular atrophy (SBMA).

[0040] In another preferred embodiment, the formulation or composition includes a carrier containing a nucleic acid molecule encoding the transaminase or deaminase or a fragment thereof.

[0041] In another preferred embodiment, the vector includes liposomes, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0042] In another preferred embodiment, the transaminase or deaminase or a fragment thereof further includes a peptide that specifically targets the cofactor or metabolic substrate.

[0043] A second aspect of the present invention provides an active ingredient combination, the active ingredient combination comprising:

[0044] (i) A first active ingredient, wherein the first active ingredient is a protein or fragment thereof selected from the group consisting of mRNA or expression vector expressing the protein:

[0045] Transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine gamma-glutamyltransferase 2 (TGM2).

[0046] Deaminases, wherein the deaminases are selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), carbamoyl-phosphate synthetase 2 (Carbamoyl-phosphate synthetase 2)-aspartate transcarbamylase and dihydroorotase fusion gene (CAD), human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpes simplex virus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia lethal factor 1. pseudomallei Burkholderia lethal factor 1 (BLF1), Shigella flexneri OspI; or combinations thereof; and

[0047] (ii) A second active ingredient, the second active ingredient being selected from the group consisting of aspartic acid, cofactors or metabolic substrates of the deaminase or transaminase, or combinations thereof.

[0048] In another preferred embodiment, the cofactor or metabolic substrate of the transaminase or deaminase includes:

[0049] Cofactors or metabolic substrates of asparagine synthase: aspartic acid, adenosine triphosphate (ATP);

[0050] Cofactor or metabolic substrate of phosphoaminotransferase: 5-phospho-alpha-D-ribose 1;

[0051] The cofactor or metabolic substrate of CTP synthase 1: uridine triphosphate (UTP);

[0052] The cofactor or metabolic substrate of CTP synthase 2: uridine triphosphate (UTP);

[0053] Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 1: D-fructose 6-phosphate;

[0054] Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 2: D-fructose 6-phosphate;

[0055] Cofactor or metabolic substrate of glutamine-dependent NAD+ synthase: deamido-NAD+;

[0056] Cofactor or metabolic substrate of GMP synthase: Xanthic acid (XMP);

[0057] The cofactor or metabolic substrate of the fusion gene (CAD) of carbamoyl synthase II-aspartate transcarbamoylase and dihydroorotase is hydrogen carbonate.

[0058] A third aspect of the invention provides the use of the active ingredient combination described in the second aspect of the invention for preparing a formulation or composition, said formulation or composition for:

[0059] (1) Convert glutamine residues in polyQ protein aggregates into glutamate residues;

[0060] (2) Promotes the degradation of polyglutamine-containing proteins or their aggregates;

[0061] (3) Reduce the proportion of polyQ aggregate-positive cells in vitro;

[0062] (4) Reduce the neurotoxicity of polyglutamine protein or its aggregates; and / or

[0063] (5) Treatment of polyglutamine diseases.

[0064] A fourth aspect of the present invention provides a pharmaceutical composition comprising: (A) the combination of active ingredients as described in the second aspect of the present invention, and (B) a pharmaceutically acceptable carrier.

[0065] In another preferred embodiment, the content of the active ingredient combination in the pharmaceutical composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the pharmaceutical composition.

[0066] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of oral preparations and injections.

[0067] In another preferred embodiment, the oral preparation is selected from the group consisting of capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, and tinctures.

[0068] In another preferred embodiment, the injectable is a liquid formulation or a lyophilized formulation.

[0069] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of oral, intravenous, intramuscular, parenteral, transdermal, intraperitoneal, or combinations thereof.

[0070] In another preferred embodiment, the preparation is a laboratory preparation.

[0071] A fifth aspect of the present invention provides a medicine box, the medicine box comprising:

[0072] (C1) A first formulation located within a first container, the first formulation comprising a first active ingredient, the first active ingredient being a protein or fragment thereof selected from the group consisting of mRNA or expression vectors expressing said protein:

[0073] Transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine gamma-glutamyltransferase 2 (TGM2).

[0074] Deaminases, wherein the deaminases are selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), carbamoyl-phosphate synthetase 2 (Carbamoyl-phosphate synthetase 2)-aspartate transcarbamylase and dihydroorotase fusion gene (CAD), human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpes simplex virus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia lethal factor 1. pseudomallei Burkholderia lethal factor 1 (BLF1), Shigella flexneri OspI; or combinations thereof;

[0075] (C2) A second formulation located within a second container, the second formulation comprising a second active ingredient selected from the group consisting of: aspartic acid, cofactors or metabolic substrates of the said deaminase or transaminase; and

[0076] (C3) Instructions for use, which state that the medicine box is used to treat polyglutamine diseases and / or delay the onset or progression of polyglutamine diseases.

[0077] A sixth aspect of the present invention provides a method for promoting the degradation of polyQ protein aggregates in vitro, comprising the steps of:

[0078] Contacting cells with the active ingredient or an expression vector that overexpresses the active ingredient promotes the degradation of the polyQ aggregates;

[0079] The active ingredient is selected from (a) transaminases and optionally cofactors or metabolic substrates of the transaminases, wherein the transaminases are selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine γ-glutamyltransferase 2 (TGM2).

[0080] (b) A deaminase and optionally a cofactor or metabolic substrate of the deaminase, wherein the deaminase is selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), a fusion gene (CAD) of carbamoyl-phosphate synthetase 2-aspartate transcarbamylase and dihydroorotase, human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpesvirus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), and Vibrio parahaemolyticus VopC. VopC), Burkholderia pseudomallei lethal factor 1 (BLF1), and Shigella flexneri OspI;

[0081] (c) A combination of components (a) and (b); and / or

[0082] (d) Aspartic acid.

[0083] In another preferred embodiment, the active ingredient is used to convert at least 1 / 30, preferably at least 1 / 10, more preferably at least 1 / 5, more preferably at least 1 / 4, more preferably at least 1 / 3, and most preferably at least 1 / 2 of the glutamine (Q) residues in the polyQ protein aggregate into glutamate (E) residues.

[0084] In another preferred embodiment, at least 1 / 30, preferably at least 1 / 10, of the glutamine (Q) residues are converted to glutamate (E) residues, thereby substantially degrading the insoluble polyQ protein aggregates.

[0085] In another preferred embodiment, the basic degradation refers to at least 60%, more preferably at least 70%, more preferably at least 80%, and more preferably at least 90% degradation of polyQ protein aggregates.

[0086] In another preferred embodiment, the cells are cells that highly express polyQ aggregates.

[0087] In another preferred embodiment, the concentration of the active ingredient is 0.1 mM to 10 mM; preferably, 0.1 mM to 1 mM; more preferably, 0.5 mM to 0.1 mM; even more preferably, 0.5 mM to 10 mM.

[0088] A seventh aspect of the present invention provides a method for treating polyglutamine disease, comprising: administering to a subject in need a therapeutically effective amount of the combination of active ingredients of the second aspect of the present invention or the pharmaceutical composition of the fifth aspect of the present invention.

[0089] In another preferred embodiment, the subjects include rodents (such as mice and rats) and primates (such as humans).

[0090] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0091] Figure 1 shows the effect of overexpression of transaminases and deaminases on the level of HTT-polyQ aggregates.

[0092] Figure 2 shows the effects of asparaginase ASNS, glutaminase GAC, and aspartic acid (ASP) on polyQ deamination.

[0093] Figure 3 shows that HTT-polyQ deamination reduces the formation of protein aggregates.

[0094] Figure 4 shows that multiple transaminases and deaminases reduce the production of HTT-polyQ protein aggregates.

[0095] Figure 5 shows the degradation effect of combined treatment with asparaginase ASNS and aspartic acid on HTT-polyQ aggregates.

[0096] Figure 6 shows that aspartic acid treatment significantly reduced the level of HTT-polyQ aggregates. Detailed Implementation

[0097] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that a series of transaminases (such as arginine synthase ASNS) can degrade polyglutamine aggregates by deaminating a small number of glutamine residues in polyglutamine (polyQ) protein, thereby reducing its neurotoxicity and treating polyglutamine-related neurodegenerative diseases. Furthermore, the cofactors or metabolic substrates (such as aspartic acid) of these deaminases or transaminases can promote their activity, thus enhancing the deamination effect on polyglutamine. Based on this discovery, the present invention was completed.

[0098] the term

[0099] As used herein, the term "first active ingredient" refers to a protein or fragment thereof selected from the group consisting of:

[0100] Transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine gamma-glutamyltransferase 2 (TGM2).

[0101] Deaminases, selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), carbamoyl-phosphate synthetase 2 (Carbamoyl-phosphate synthetase 2)-aspartate transcarbamylase and dihydroorotase fusion gene (CAD), human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpes simplex virus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, and Burkholderia melioides lethal factor 1. pseudomallei Burkholderia lethal factor 1 (BLF1), Shigella flexneri OspI, or combinations thereof.

[0102] As used herein, the term "second active ingredient" refers to aspartic acid, a cofactor or metabolic substrate of the deaminase or transaminase, or a combination thereof.

[0103] As used herein, the terms “combination of active ingredients of the present invention”, “combination of the present invention”, and “combination of pharmaceutical ingredients of the present invention” are used interchangeably to refer to the combination of the first and second active ingredients described above.

[0104] As used herein, the term "pharmaceutical composition of the present invention" refers to a composition comprising or containing a first active ingredient (or a first formulation containing the first active ingredient) and a second active ingredient (or a second formulation containing the second active ingredient), wherein the first and second formulations may be the same or different formulations. Furthermore, the first and second formulations may be the same formulation or separate formulations.

[0105] Polyglutamine disease

[0106] Polyglutamine (PolyQ) disorders are characterized by the aberrant amplification of the cytosine-adenine-guanine (CAG) trinucleotide repeat, resulting in proteins with elongated polyglutamine chains. Although a rare disease, with an average incidence of 1-10 cases per 100,000 people, polyglutamine disorders constitute the largest group of monogenic neurodegenerative diseases. These diseases are incurable and have no preventative measures, making treatment extremely complex, and their hereditary nature can severely impact the entire family.

[0107] Known polyglutamine disorders include, but are not limited to: Huntington's disease (HD), six types of spinocerebellar ataxia (SCA) including types I, II, III, VI, VII, and XVII; dentate nucleus-rubella-leucocerebellar ataxia (DRPLA); spinal bulbar muscular atrophy (SBMA); etc.

[0108] Huntington's disease:

[0109] Huntington's disease (HD) is a rare autosomal dominant inherited neurodegenerative disorder. The cause of Huntington's disease is an abnormal amplification of the CAG triplet repeat sequence in exon 1 of the huntingtin gene (HTT) on chromosome 4. In 1993, the international Huntington's disease collaborative research group cloned the disease-causing gene IT15, also known as the HTT gene, located on the upper part of chromosome 4. Its metabolite, huntingtin protein, has a relative molecular mass of 350 kDa and is composed of 3144 amino acids. Hunttin protein is widely expressed in various organs throughout the body, including the central nervous system. Its normal function is not fully understood, but it may be related to nervous system development, endocytosis and secretion, and inhibition of apoptosis. The PolyQ portion of huntingtin protein is encoded by a repeating CAG trinucleotide repeat sequence. In normal individuals, this repetitive sequence has 6-35 repeats. Abnormal huntingtin proteins with more than 40 repeats tend to stick together and aggregate, ultimately leading to nerve cell death and motor symptoms. Mutant HTT (mHTT) aggregates in the cell nucleus to form inclusion bodies, which is the core pathophysiological mechanism of HD.

[0110] HTT protein:

[0111] The human HTT gene is located on the short arm of chromosome 4 and has up to 67 exons. The pathogenic site for Huntington's disease is in the first exon. Mutations in this gene are mostly of three types: haplotype A1, A2, and A3. Important regions of the HTT protein include the N-terminal polyglutamine region and polyproline region; and the three HEAT regions containing the most amino acids (this region was first discovered in four proteins). HTT has recognition sites for the proteasome, caspase, and calpain. Under normal circumstances, HTT can be hydrolyzed into two segments by caspase, and the two segments can bind together to perform their functions. However, in the pathological condition of HD, HTT expression not only increases, but its free N-terminal product is more prone to aggregation, and the C-terminus also produces certain toxicity.

[0112] Asparagine synthase (ASNS):

[0113] The asparagine synthase gene is located on human chromosome 7, specifically in region 7q21.3, with a genome sequence length of 35kb and containing 13 exons. ASNS is a member of the aminotransferase family, primarily located in the cytoplasm. Its main biological function is to catalyze the conversion of aspartate (Asp) to asparagine (Asn) under the influence of ATP. Congenital defects in asparagine biosynthesis lead to severe neurological disorders characterized by microcephaly, severely delayed psychomotor development, progressive encephalopathy, cortical atrophy, and seizures or hyperflexion.

[0114] Phosphoribose pyrophosphate aminoacyltransferase (PPAT):

[0115] PPAT is a key rate-limiting enzyme in purine biosynthesis. It catalyzes the first step in the de novo purine nucleotide biosynthesis pathway, transferring γ-nitrogen from glutamine to 5-phosphoribosyl 1-pyrophosphate (PRPP).

[0116] Cytidine triphosphate synthase 1 (CTPS1) / Cytidine triphosphate synthase 2 (CTPS2):

[0117] Cytidine triphosphate synthase (CTPS) is the rate-limiting enzyme in the de novo synthesis of cytosine nucleotides and the uridine reuptake synthesis process. It is mainly composed of a glutaminase domain and an amide oligosaccharide domain. Currently, two subtypes have been identified in humans, encoded by the CTPS1 and CTPS2 genes, respectively. CTP is the final product of pyrimidine synthesis in which CTPS participates. It is a precursor to RNA and DNA synthesis and participates in various metabolic processes, including phospholipid synthesis and intracellular signal transduction pathways.

[0118] Glutamine-6-phosphate fructose aminotransferase 1 (GFPT1) / Glutamine-6-phosphate fructose aminotransferase 2 (GFPT2):

[0119] Glutamine-6-phosphate fructose aminotransferase (HBP), encoded by homologs GFPT1 and GFPT2, is the rate-limiting enzyme of the hexosamine pathway, responsible for glycosylation. This gene encodes the first rate-limiting enzyme in the hexosamine pathway and controls the flow of glucose into it. The product of this gene catalyzes the formation of glucosamine 6-phosphate. It controls the flux of glucose into the hexosamine pathway. It may be involved in regulating the availability of N- and O-linked glycosylation precursors in proteins. It regulates the circadian rhythm expression of the clock genes BMAL1 and CRY1. It plays a role in fine-tuning metabolic fluctuations in cytoplasmic UDP-GlcNAc and its influence on hyaluronic acid synthesis during tissue remodeling.

[0120] Glutamine-dependent NAD + Synthesizer (NADSYN1):

[0121] Nicotinamide adenine dinucleotide synthetase 1 (NADSYN1) is an important enzyme for the synthesis of nicotinamide adenine dinucleotide (NAD). NAD is a coenzyme for more than 400 cellular redox reactions in the human body, and also participates in processes such as mitochondrial metabolism, DNA repair, cell division, inflammation, and immune response.

[0122] GMP synthase (GMPS):

[0123] Guanosyl monophosphate synthase (GMPS) catalyzes the final step in the guanylate branch of purine biosynthesis. In this reaction, glutamine is hydrolyzed, and the resulting ammonia is incorporated into xanthan monophosphate (XMP) to form GMP. Therefore, GMPS plays an important role in nucleotide biosynthesis in normal cell proliferation and tumorigenesis. In de novo purine nucleotide synthesis, IMP is a branching metabolite that differentiates into guanine or adenine nucleotides. In the guanine nucleotide pathway, two enzymes are involved in the conversion of IMP to GMP: IMP dehydrogenase (IMPD1), which catalyzes the oxidation of IMP to XMP, and GMP synthase, which catalyzes the amination of XMP to GMP.

[0124] Asparaginase (ASRGL1):

[0125] Asparaginase 1 (ASRGL1) is a member of the N-terminal nucleophilic (Ntn) hydrolase superfamily. ASRGL1 primarily catalyzes the hydrolysis of isoaspartate dipeptides into L-aspartate and ammonia, playing a crucial role in various metabolic activities. Located at position 11q12.3 on human chromosome, it contains 307 amino acid residues and has a molecular weight of 31.9 kDa. L-aspartate acts as an excitatory neurotransmitter in certain brain regions; therefore, impaired L-aspartate hydrolysis may lead to neurological dysfunction. Isoaspartate dipeptides are a common source of non-enzymatic protein damage. Proteins containing isoaspartate dipeptides have a significant impact on substrate recognition and conversion of various proteases, and in some cases even act as protease inhibitors.

[0126] Aspartic acid

[0127] Aspartic acid, also known as aspartic acid, is an α-amino acid. The levorotatory isomer of aspartic acid is one of the 20 protein amino acids. Aspartic acid is widely present in biosynthesis. It is a precursor in the synthesis of amino acids such as lysine, threonine, isoleucine, and methionine, as well as purine and pyrimidine bases. It can serve as a precursor for potassium (K) in the body. + Mg 2+ It acts as an ion carrier, delivering electrolytes to the myocardium, thereby improving myocardial contractility and reducing oxygen consumption. This provides protection for the myocardium during coronary circulatory disorders and hypoxia. It participates in the ornithine cycle, promoting the formation of urea from ammonia and carbon dioxide, reducing blood nitrogen and carbon dioxide levels, enhancing liver function, and relieving fatigue.

[0128] For mammals, aspartic acid is non-essential, as it can be produced from oxaloacetate via transamination. For plants and microorganisms, aspartic acid is a precursor to several amino acids, including the four essential ones: methionine, threonine, isoleucine, and lysine. The conversion from aspartic acid to these amino acids begins with the conversion of aspartic acid to its "semialdehyde." Asparagine is produced from aspartic acid via transamination. In the food industry, aspartic acid is a good nutritional supplement added to various soft drinks; it is also a major raw material for the sweetener aspartame (aspartame-asparagine methyl ester).

[0129] Combination of active ingredients, pharmaceutical composition

[0130] This invention provides an active ingredient combination comprising a class of deaminases or transaminases and their cofactors or metabolic substrates, which can be used to prepare drugs for treating polyglutamine diseases such as Huntington's disease.

[0131] Based on the above-mentioned components, the present invention provides a pharmaceutical composition that can be used for:

[0132] (1) Convert glutamine residues in polyQ protein aggregates into glutamate residues;

[0133] (2) Promotes the degradation of polyglutamine-containing proteins or their aggregates;

[0134] (3) Reduce the proportion of polyQ aggregate-positive cells in vitro;

[0135] (4) Reduce the neurotoxicity of polyglutamine protein or its aggregates; and / or

[0136] (5) Treatment of polyglutamine diseases.

[0137] The present invention also provides a pharmaceutical composition comprising a combination of active ingredients within a safe and effective range, and a pharmaceutically acceptable carrier.

[0138] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, etc.

[0139] There are no particular limitations on the administration of the active ingredient combination or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0140] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0141] When using pharmaceutical preparations, safe and effective amounts of active ingredients are combined and administered to mammals.

[0142] It should be understood that the effective amount of the active ingredient described in this invention may vary depending on the administration method and the severity of the disease. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, and half-life; disease severity; patient weight; patient immune status; and route of administration.

[0143] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0144] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0145] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0146] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0147] The main advantages of this invention include:

[0148] (1) This invention has discovered a series of transaminases or deaminases that can perform non-classical functions, converting a small amount (about 1 / 30) of glutamine residues in polyglutamine protein into glutamic acid, thereby degrading polyglutamine protein or its aggregates, thus fundamentally reversing the occurrence and development of polyglutamine diseases.

[0149] (2) The present invention uses a series of transaminases or deaminases and their cofactors or metabolic substrates in combination to achieve synergistic therapeutic effects.

[0150] (3) The active ingredients transaminase or deaminase used in this invention are delivered to the relevant concurrent areas using technologies such as adeno-associated virus (AAV) or liposome encapsulation, which is safe and efficient; the cofactor or metabolic substrate of the other active ingredient transaminase or deaminase used in this invention are metabolites that are naturally present in the human body and have high safety for the human body.

[0151] (4) This invention is applicable to a variety of glutamine diseases, such as Huntington's disease (HD), six types of spinocerebellar ataxia (SCA) including type I, II, III, VI, VII and XVII; dentate nucleus-rubella-leucocerebellar ataxia (DRPLA); and spinal bulbar muscular atrophy (SBMA), etc., and can achieve the goal of "treating different diseases with the same method".

[0152] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0153] Example 1. Overexpression of transaminases and deaminases reduces the level of HTT-polyQ aggregates.

[0154] First, SY-5Y neural cells stably expressing HTT(1-17)-polyQ(91)-GFP were constructed. Then, the cells were transiently transfected with plasmids overexpressing the corresponding transaminase or deaminase or their empty vector control. After 24 hours, the cells were collected, and the soluble and insoluble fractions were separated for immunoblotting experiments to detect the level of insoluble polyQ protein aggregates.

[0155] As shown in Figure 1, overexpression of ASNS, ASRGL1, PPAT, CTPS1, CTPS2, GFPT1, GFPT2, GMPS, NADSYN1, GLS, and GAC significantly reduced the level of insoluble polyQ protein aggregates. Furthermore, overexpression of some proteins caused an upward shift of the polyQ band, suggesting a deamination reaction. Because the charge of the deaminated protein changes, the protein aggregates in different treatment systems exhibited varying degrees of shift.

[0156] Example 2. In vitro catalytic deamination of polyQ by asparaginase ASNS and glutaminase GAC

[0157] The purified ASNS or GAC protein and polyQ peptide, along with aspartic acid, were reacted at 30°C for 1 hour in a buffer containing MgCl2 and ATP. The samples were then analyzed by dot blot using the corresponding antibodies.

[0158] As shown in Figure 2, both ASNS and GAC can catalyze the conversion of polyQ to polyE, and aspartic acid (ASP) can further enhance the activity of the deamination reaction. This indicates that both ASNS and GAC can catalyze the deamination of polyQ in vitro, and that the metabolic substrate of ASNS, aspartic acid (ASP), can accelerate the reaction rate.

[0159] Example 3. HTT-polyQ deamination reduces protein aggregate formation.

[0160] Transient transfection of SY-5Y cells with HTT(1-17)-polyQ(91)-GFP or mutant plasmids of which 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, and 1 / 30 of the glutamine (Q) residues were converted to glutamate (E) residues, respectively, followed by cell fixation and immunofluorescence assays 24 h later.

[0161] As shown in Figure 3, HTT(1-17)-polyQ(91) can form obvious aggregates in cells, while HTT(1-17)-polyQ proteins with Q to E mutations at ratios of 1 / 2, 1 / 3, 1 / 4, 1 / 5, and 1 / 10 do not form aggregates. HTT(1-17)-polyQ proteins with Q to E mutations at ratios of 1 / 30 still form aggregates, but the proportion of aggregates formed is significantly reduced. These findings indicate that deamination of the Q to E groups in HTT-polyQ proteins can prevent or reduce the formation of protein aggregates.

[0162] Example 4. Transaminases and deaminases reduce the production of HTT-polyQ protein aggregates.

[0163] SY-5Y cells stably expressing HTT(1-17)-polyQ(91)-GFP were constructed, and then the cells were transiently transfected with a plasmid overexpressing ASNS or its empty vector control. After 24 hours, the cells were fixed and immunofluorescence staining was performed, and the proportion of cells with polyQ aggregates was counted.

[0164] As shown in Figure 4, the proportion of polyQ aggregates in SY-5Y cells transfected with the empty vector control was approximately 35%, while in SY-5Y cells overexpressing ASNS protein, the proportion of polyQ aggregates was approximately 13%. This indicates that ASNS overexpression significantly reduced polyQ aggregate formation. As shown in Table 1, overexpression of other transaminases or deaminases also significantly reduced polyQ aggregate formation.

[0165] Table 1

[0166] Example 5. Combined treatment with asparaginase ASNS and aspartic acid further reduced HTT-polyQ aggregates.

[0167] In SY-5Y cells stably expressing HTT(1-17)-polyQ(91)-GFP, a plasmid overexpressing ASNS or its empty vector control was transiently transfected, and different concentrations of aspartic acid were added to the cell culture medium. After 24 h, cells were collected for immunoblotting to detect the levels of soluble polyQ protein and insoluble HTT-polyQ aggregates.

[0168] As shown in Figure 5, compared with the control plasmid, ASNS overexpression or aspartic acid treatment alone can significantly reduce the level of insoluble HTT-polyQ aggregates. The combined treatment of the two can further exert the deamination effect of ASNS, which can further reduce the level of insoluble HTT-polyQ aggregates.

[0169] Example 6. Aspartic acid treatment significantly reduced the level of HTT-polyQ aggregates.

[0170] In SY-5Y cells stably expressing HTT(1-17)-polyQ(91)-GFP, different concentrations of aspartic acid were added to the cell culture medium. After 24 hours, the cells were fixed and immunofluorescence was performed.

[0171] As shown in Figure 6, compared with the control group, aspartic acid treatment can significantly reduce the level of insoluble HTT-polyQ aggregates.

[0172] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient, characterized in that, Used to prepare a formulation or composition, wherein the active ingredient is selected from: (a) A transaminase and optionally a cofactor or metabolic substrate of the transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), protein-glutamine γ-glutamyltransferase 2 (TGM2); or combinations thereof; (b) A deaminase and optionally a cofactor or metabolic substrate of the deaminase, wherein the deaminase is selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), a fusion gene (CAD) of carbamoyl-phosphate synthetase 2-aspartate transcarbamylase and dihydroorotase, human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpesvirus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), and Vibrio parahaemolyticus VopC. VopC), Burkholderia pseudomallei lethal factor 1 (BLF1), and Shigella flexneri OspI; (c) A combination of components (a) and (b); and / or (d) Aspartic acid; And the formulation or composition is intended for use selected from the group consisting of: (1) Convert some glutamine residues in polyQ protein aggregates into glutamate residues; (2) Promotes the degradation of polyglutamine-containing proteins or their aggregates; (3) Reduce the proportion of polyQ aggregate-positive cells in vitro; (4) Reduce the neurotoxicity of polyglutamine protein or its aggregates; and / or (5) Treatment of polyglutamine diseases.

2. The use as described in claim 1, characterized in that, The cofactors or metabolic substrates of the transaminase or deaminase include: Cofactors or metabolic substrates of asparagine synthase: aspartic acid, adenosine triphosphate (ATP); Cofactor or metabolic substrate of phosphoaminotransferase: 5-phospho-alpha-D-ribose 1; The cofactor or metabolic substrate of CTP synthase 1: uridine triphosphate (UTP); The cofactor or metabolic substrate of CTP synthase 2: uridine triphosphate (UTP); Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 1: D-fructose 6-phosphate; Cofactor or metabolic substrate of glutamine-6-phosphate fructose aminotransferase 2: D-fructose 6-phosphate; Cofactor or metabolic substrate of glutamine-dependent NAD+ synthase: deamido-NAD+; Cofactor or metabolic substrate of GMP synthase: Xanthic acid (XMP); The cofactor or metabolic substrate of the fusion gene (CAD) of carbamoyl synthase II-aspartate transcarbamoylase and dihydroorotase is hydrogen carbonate.

3. The use as described in claim 1, characterized in that, The polyglutamine-containing protein is selected from the following group: Huntingtin (HTT) protein, TATA box-binding protein (TBP), atrophin-1, and androgen receptor (AR).

4. The use as described in claim 1, characterized in that, The polyglutamine diseases mentioned are selected from the following groups: Huntington's disease (HD), six types of spinocerebellar ataxia (SCA) including types I, II, III, VI, VII and XVII; dentate nucleus-rubella-leucocerebellar Lewy body atrophy (DRPLA); and spinal bulbar muscular atrophy (SBMA).

5. The use as described in claim 1, characterized in that, The formulation or composition includes a carrier containing a nucleic acid molecule encoding the transaminase or deaminase or a fragment thereof.

6. A combination of active ingredients, characterized in that, The combination of active ingredients includes: (i) A first active ingredient, wherein the first active ingredient is a protein or fragment thereof selected from the group consisting of mRNA or expression vector expressing the protein: Transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine gamma-glutamyltransferase 2 (TGM2). Deaminases, wherein the deaminases are selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), carbamoyl-phosphate synthetase 2 (Carbamoyl-phosphate synthetase 2)-aspartate transcarbamylase and dihydroorotase fusion gene (CAD), human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpes simplex virus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia lethal factor 1. pseudomallei Burkholderia lethal factor 1 (BLF1), Shigella flexneri OspI; or combinations thereof; and (ii) A second active ingredient, the second active ingredient being selected from the group consisting of aspartic acid, cofactors or metabolic substrates of the deaminase or transaminase, or combinations thereof.

7. Use of the active ingredient combination as described in claim 6, for the preparation of a formulation or composition, said formulation or composition being used for: (1) Convert glutamine residues in polyQ protein aggregates into glutamate residues; (2) Promotes the degradation of polyglutamine-containing proteins or their aggregates; (3) Reduce the proportion of polyQ aggregate-positive cells in vitro; (4) Reduce the neurotoxicity of polyglutamine protein or its aggregates; and / or (5) Treatment of polyglutamine diseases.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (A) the active ingredient combination as described in claim 6, and (B) a pharmaceutically acceptable carrier.

9. A medicine box, characterized in that, The medicine box includes: (C1) A first formulation located within a first container, the first formulation comprising a first active ingredient, the first active ingredient being a protein or fragment thereof selected from the group consisting of mRNA or expression vectors expressing said protein: Transaminase, wherein the transaminase is selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine gamma-glutamyltransferase 2 (TGM2). Deaminases, wherein the deaminases are selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), carbamoyl-phosphate synthetase 2 (Carbamoyl-phosphate synthetase 2)-aspartate transcarbamylase and dihydroorotase fusion gene (CAD), human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpes simplex virus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia lethal factor 1. pseudomallei Burkholderia lethal factor 1 (BLF1), Shigella flexneri OspI; or combinations thereof; (C2) A second formulation located within a second container, the second formulation comprising a second active ingredient selected from the group consisting of: aspartic acid, cofactors or metabolic substrates of the said deaminase or transaminase; and (C3) Instructions for use, which state that the medicine box is used to treat polyglutamine diseases and / or delay the onset or progression of polyglutamine diseases.

10. A method for promoting the degradation of polyQ protein aggregates in vitro, comprising the steps of: Contacting cells with the active ingredient or an expression vector that overexpresses the active ingredient promotes the degradation of the polyQ aggregates; The active ingredient is selected from (a) transaminases and optionally cofactors or metabolic substrates of the transaminases, wherein the transaminases are selected from the group consisting of: asparagine synthase (ASNS), phosphoaminotransferase (PPAT), CTP synthase 1 (CTPS1), CTP synthase 2 (CTPS2), glutamine-6-phosphofructoaminotransferase 1 (GFPT1), glutamine-6-phosphofructoaminotransferase 2 (GFPT2), glutamine-dependent NAD+ synthase (NADSYN1), GMP synthase (GMPS), formylglycine aminonucleotide synthase (PFAS), and protein-glutamine γ-glutamyltransferase 2 (TGM2). (b) A deaminase and optionally a cofactor or metabolic substrate of the deaminase, wherein the deaminase is selected from the group consisting of: asparaginase (ASRGL1), glutaminase (GLS, GAC), a fusion gene (CAD) of carbamoyl-phosphate synthetase 2-aspartate transcarbamylase and dihydroorotase, human N-terminal glutamine aminohydrolase (NTAQ1), herpes simplex virus UL37 protein (HSV UL37), herpesvirus ORF75 / ORF75c protein, E. coli CNFs, E. coli cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), and Vibrio parahaemolyticus VopC. VopC), Burkholderia pseudomallei lethal factor 1 (BLF1), and Shigella flexneri OspI; (c) A combination of components (a) and (b); and / or (d) Aspartic acid.

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