A novel treatment for diseases carrying aspartate residue mutations

Abstract

The present application provides a novel treatment method for diseases carrying aspartate residue mutations, specifically, the present application directly aspartate deaminates proteins carrying aspartate residue mutations (such as KRAS G12D protein) by asparagine synthetase (ASNS), effectively reduces the expression amount of proteins carrying aspartate residue mutations, and further inhibits downstream signaling pathways (such as ERK / AKT pathway), thereby treating diseases carrying aspartate residue mutations. In addition, the cofactor (such as glutamine) of ASNS can promote the activity of ASNS and enhance the above deamination effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to a novel treatment method for diseases involving mutations in aspartic acid residues. Background Technology

[0002] KRAS G12D is one of the most common oncogenic mutations in the KRAS gene. It is caused by a missense mutation at codon 12 (c.35G>A), resulting in the substitution of glycine (G) for aspartic acid (D) (p.Gly12Asp). This mutation is located in the P-loop domain of the KRAS protein and disrupts GTP hydrolysis activity, keeping the protein in a persistently activated state. This leads to abnormal activation of downstream signaling pathways such as MAPK and PI3K / AKT, promoting tumor cell proliferation, invasion, and drug resistance. G12D mutations are prevalent in pancreatic ductal adenocarcinoma (approximately 40%), colorectal cancer (approximately 10-15%), and non-small cell lung cancer (approximately 4%), and are particularly closely associated with pancreatic cancer progression. Clinical studies have shown that G12D mutations can lead to primary resistance to anti-EGFR monoclonal antibodies (such as cetuximab), and traditional targeted therapies are limited.

[0003] KRAS G13D is a common oncogenic mutation in the KRAS gene, specifically a missense mutation at codon 13 (glycine is replaced by aspartic acid, p.Gly13Asp). This mutation leads to abnormal GTPase activity in the KRAS protein, preventing it from effectively hydrolyzing GTP to GDP, thus keeping it in an activated state. This abnormally activates downstream signaling pathways such as MAPK and PI3K / AKT, promoting cell proliferation, survival, and tumor development. Clinically, it is most commonly seen in colorectal cancer (approximately 8-10%), pancreatic ductal adenocarcinoma (approximately 14%), and non-small cell lung cancer (approximately 4%). Unlike some KRAS mutations, studies suggest that the G13D mutation in colorectal cancer may retain some sensitivity to anti-EGFR monoclonal antibody (such as cetuximab) therapy, but the evidence remains controversial.

[0004] BRAF V600D is an oncogenic mutation in the BRAF gene, a rare subtype within the BRAF V600 mutation family. It is caused by a missense mutation at codon 600 (c.1799T>A) resulting in the substitution of valine (V) for aspartic acid (D) (p.Val600Asp). This mutation is located in the activation loop of the BRAF kinase domain, stabilizing the kinase's activated conformation and continuously activating the downstream MAPK / ERK signaling pathway, driving tumor cell proliferation, survival, and metastasis. BRAF V600D is commonly found in melanoma (approximately 1-2%), colorectal cancer (<1%), and papillary thyroid carcinoma. Its clinical features are similar to the classic V600E mutation, but its invasiveness may be lower compared to V600K / R. In terms of treatment, BRAF V600D is less sensitive to BRAF single-target inhibitors (such as vemurafenib) than V600E, but combining it with MEK inhibitors (such as dabrafenib + trametinib) may partially overcome drug resistance.

[0005] KCNH2 N588D is a point mutation known to be associated with cardiac arrhythmias. It refers to the replacement of asparagine (N) with aspartic acid (D) at amino acid position 588 of the potassium channel protein (hERG) encoded by the KCNH2 gene. This mutation is located in the S5-P loop region of the channel and is a key structure regulating the inactivation process. The N588D mutation significantly weakens the rapid inactivation capacity of the hERG channel, leading to accelerated potassium ion efflux, premature repolarization of the myocardial action potential, and thus shortening the QT interval on the electrocardiogram. Clinically, this mutation is closely associated with short QT syndrome (SQTS) and is one of the typical pathogenic mutations of SQTS1, commonly found in individuals with a family history of sudden death or recurrent syncope.

[0006] CFTR G551D is a common pathogenic mutation in the CFTR gene, in which the amino acid at position 551 is replaced by aspartic acid (D) instead of glycine (G). The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene encodes a chloride ion channel widely distributed on the cell membranes of epithelial cells in the lungs, pancreas, and intestines, and is crucial for maintaining electrolyte and fluid balance. The G551D mutation belongs to class III mutations, and its main pathological mechanism is that although the CFTR channel can be correctly located on the cell membrane, its opening efficiency is significantly reduced, leading to chloride ion transport impairment. CFTR G551D is one of the most common non-F508del mutations in cystic fibrosis (CF), accounting for approximately 4-5% of all CF cases, especially prevalent in European and American populations. Carriers of this mutation often present with typical CF clinical symptoms, such as recurrent lung infections, pancreatic insufficiency, and malabsorption.

[0007] All of the aforementioned diseases involve mutations in the aspartic acid residues of certain genes, such as KRAS G12D. Therefore, there is an urgent need to develop a drug that can inhibit mutations in the aspartic acid residues of genes, which may hopefully treat various diseases carrying this mutation. Summary of the Invention

[0008] The purpose of this invention is to provide a drug that inhibits mutations in aspartic acid residues in genes.

[0009] In a first aspect of the invention, there is provided the use of a deaminase or transaminase and optionally a cofactor of said deaminase or transaminase for the preparation of a drug / formulation having uses selected from the group consisting of:

[0010] a. Directly deaminate the aspartic acid residue in the protein carrying the mutation, thereby converting the aspartic acid at the mutation site into asparagine;

[0011] b. Catalyzing the deamination of proteins or peptides carrying mutations in aspartic acid residues in vitro;

[0012] c. Inhibit tumor growth; and / or

[0013] d. To treat diseases carrying mutations in aspartic acid residues or to improve the sensitivity of said diseases to immunotherapy or conventional treatment.

[0014] In another preferred embodiment, the deaminase or transaminase is an exogenous deaminase or transaminase.

[0015] In another preferred embodiment, the deaminase or transaminase is asparagine synthase (ASNS).

[0016] In another preferred embodiment, the cofactor is glutamine.

[0017] In another preferred embodiment, the protein carrying the aspartic acid residue mutation is the KRAS G12D protein, and ASNS binds to the KRASD12 site via ASNS K378.

[0018] In another preferred embodiment, the protein carrying the aspartic acid residue mutation is selected from the group consisting of: Kirsten rat sarcoma virus oncogene homolog (KRAS), Harvey rat sarcoma virus oncogene homolog (HRAS), neuroblastoma Ras virus oncogene homolog (NRAS), B-Raf proto-oncogene serine / threonine protein kinase (BRAF), cystic fibrosis transmembrane conductance regulator (CFTR) protein, potassium voltage-gated channel subfamily H member 2 (KCNH2), glucocerebroside lipase (GBA), β-globin, phenylalanine hydroxylase (PAH), fibrin-1 (FBN1), dystrophin (DMD), type I collagen α1 chain (COL1A1), or combinations thereof.

[0019] In another preferred embodiment, 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), or protein-glutamine γ-glutamyltransferase 2 (TGM2).

[0020] In another preferred embodiment, the deaminase is 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), HSV UL37, HSV ORF75 / ORF75c, E. coli CNFs, E. coli Cycle-inhibiting factors (Cifs), Pasteurella multocida Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia pseudomallei Burkholderialethal factor 1 (BLF1), or Shigella flexneri OspI.

[0021] In another preferred embodiment, the cofactor is selected from the group consisting of: glutamine, aspartic acid, 5-phospho-alpha-D-ribose 1-diphosphate, D-fructose 6-phosphate, and deamido-NAD. + , or combinations thereof.

[0022] In another preferred embodiment, glutamine promotes the deamination of exogenous ASNS.

[0023] In another preferred embodiment, glutamine promotes the deamination of endogenous ASNS.

[0024] In another preferred embodiment, ASNS is overexpressed intracellularly by introducing an expression plasmid of ASNS into the cell.

[0025] In another preferred embodiment, the disease carrying the aspartate residue mutation is selected from the group consisting of: pancreatic cancer, pancreatic ductal carcinoma, colorectal cancer, non-small cell lung cancer, lung cancer, melanoma, papillary thyroid carcinoma, cystic fibrosis, hereditary arrhythmias (such as long QT syndrome), lysosomal storage diseases (such as Gaucher disease), sickle cell anemia, phenylketonuria, Marfan syndrome, dystrophinosis, osteogenesis imperfecta, or combinations thereof.

[0026] In another preferred embodiment, the immunotherapy is selected from the group consisting of T-cell vaccines, tumor vaccines, immune checkpoint inhibitors, or combinations thereof.

[0027] In another preferred embodiment, the conventional treatment is selected from the group consisting of targeted therapy, chemotherapy, radiotherapy, or combinations thereof.

[0028] In a second aspect of the invention, an active ingredient combination is provided, the active ingredient combination comprising:

[0029] (i) a first active ingredient, wherein the first active ingredient is a deaminase or transaminase as described in the first aspect of the present invention, or an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase; and

[0030] Optional (ii) a second active ingredient, wherein the second active ingredient is a cofactor of the deaminase or transaminase.

[0031] In another preferred embodiment, the combination of active ingredients is asparagine synthase (ASNS) and glutamine.

[0032] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0033] (i) The first active ingredient is the deaminase or transaminase described in the first aspect of the present invention, or an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase;

[0034] Optional (ii) a second active ingredient, wherein the second active ingredient is a cofactor of the deaminase or transaminase; and

[0035] (iii) Pharmaceutically acceptable carriers.

[0036] In another preferred embodiment, the pharmaceutical composition is ASNS, glutamine, and a pharmaceutically acceptable carrier.

[0037] In a fourth aspect of the invention, a kit is provided, the kit comprising:

[0038] A first container and a first reagent within the container, wherein the first reagent is a deaminase or transaminase as described in the first aspect of the present invention, or an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase; and

[0039] An optional second container and a second reagent within the container, wherein the second reagent is a cofactor of the deaminase or transaminase.

[0040] In another preferred embodiment, the first reagent is ASNS and the second reagent is glutamine.

[0041] In another preferred embodiment, the kit also includes instructions for using the reagents.

[0042] In a fifth aspect of the invention, a method is provided for deamination of a protein or polypeptide containing a mutation of an aspartic residue in vitro, the method comprising the steps of:

[0043] 1) Co-incubate a protein or polypeptide containing an aspartic residue mutation with a reaction system containing the deaminase or transaminase described in the first aspect of the present invention, or an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase, thereby obtaining a protein or polypeptide with an aspartic residue mutation to asparagine.

[0044] In another preferred embodiment, the reaction system further includes MgCl2 and ATP.

[0045] In another preferred embodiment, the reaction system further includes a reaction buffer.

[0046] 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

[0047] Figure 1 The results showed that asparagine synthase (ASNS) and glutamine inhibited the expression of G12D in pancreatic cancer cells.

[0048] Figure 2 ASNS was shown to catalyze the deamination of KRAS G12D in vitro.

[0049] Figure 3 The study showed that glutamine promotes KRASD12N expression and inhibits ERK phosphorylation in vivo.

[0050] Figure 4 The results show that ASNS K378 and KRASD12 are the binding sites of ASNS and KRAS G12D proteins.

[0051] Figure 5 The study showed that glutamine can inhibit tumor growth in nude mice inoculated with AsPC-1 (human metastatic pancreatic adenocarcinoma cells). Detailed Implementation

[0052] Through extensive and in-depth research, the inventors have developed a novel treatment method for diseases involving aspartate residue mutations. This invention utilizes asparagine synthase (ASNS) to directly deaminate proteins carrying aspartate residue mutations (e.g., KRAS G12D protein), effectively reducing the expression level of these proteins and inhibiting downstream signaling pathways (e.g., the ERK / AKT pathway), thereby suppressing tumor growth. Furthermore, glutamine enhances the deamination activity of ASNS. This invention is based on these findings.

[0053] the term

[0054] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0055] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0056] Asparagine synthase (ASNS)

[0057] 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, and consists of two domains: the N-terminus catalyzes the hydrolysis of glutamine, and the C-terminus catalyzes the reaction of aspartate (Asp) carboxylate with adenine triphosphate (ATP) to produce asparagine. The main function of ASNS is to catalyze the conversion of Asp to asparagine (Asn) in an ATP-dependent manner, participating in cellular amino acid and energy metabolism. 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.

[0058] This invention discovers that ASNS can also perform non-classical functions, deaminating protein aspartic acid residues (such as the KRAS G12D mutation), thereby effectively reducing the expression level of mutant aspartic acid residues, and thus inhibiting downstream pathways (such as the ERK / AKT pathway), and treating diseases carrying aspartic acid residue mutations.

[0059] Glutamine

[0060] Glutamine metabolism is a crucial component of cellular life activities, playing a particularly vital role in cell growth and proliferation. Tumor cells differ from normal human cells in their metabolism. While glycolysis is inhibited in normal cells under aerobic conditions, tumor cells, under sufficient oxygen, preferentially utilize glucose for glycolysis, producing large amounts of lactic acid with limited energy production. Consequently, cancer patients actively utilize glutamine to replenish the tricarboxylic acid cycle, providing energy for cell growth and proliferation. Currently, the specific role of glutamine in tumor cell growth, differentiation, and proliferation remains unclear. However, most researchers believe that exogenous glutamine supplementation is unlikely to lead to tumor growth, local invasion, or metastasis. Therefore, glutamine supplementation for cancer patients is especially important.

[0061] In this invention, glutamine treatment significantly inhibited the intracellular expression level of KRAS G12D protein in Aspc-1 pancreatic cancer cells with KRAS G12D mutations, and also suppressed the expression of phosphorylated ERK. Furthermore, glutamine also helped promote the catalytic function of ASNS and reduced the expression of proteins with aspartic acid residue mutations.

[0062] Kirsten rat sarcoma virus oncogene homolog (KRAS)

[0063] Humans have three RAS genes—KRAS, NRAS, and HRAS—that encode four different RAS proteins. These proteins belong to the small GTPases protein family and are involved in cell signaling, functioning as "molecular switches." Activating mutations in RAS genes are among the most common oncogenic drivers in human cancers. KRAS gene mutations are the most frequent, accounting for over 20% of all tumor mutations. The most common mutations are at the p.G12D and p.G13D sites. KRAS gene mutations are closely related to the development of lung cancer, pancreatic cancer, and colorectal cancer, and are a strong negative predictor of response to anti-EGFR therapy.

[0064] Serine / threonine protein kinase B-raf gene (BRAF)

[0065] BRAF, a member of the RAF family, encodes a serine / threonine protein kinase that plays a crucial role in the MAPK / ERK signaling pathway, which regulates cell growth, proliferation, and survival. Mutations in BRAF (such as the common V600E mutation) lead to abnormal and persistent activation of this pathway, promoting tumorigenesis. BRAF mutations are associated with various cancers, including melanoma, colorectal cancer, and thyroid cancer. Targeted therapies against mutated BRAF (such as vemurafenib and dabrafenib) significantly improve patient outcomes by inhibiting kinase activity; however, drug resistance has spurred the development of combination therapies (such as BRAF / MEK dual inhibitors). Currently, BRAF mutation detection has become an important biomarker for the diagnosis and targeted therapy of related cancers.

[0066] Potassium voltage-gated channel subfamily H member 2 (KCNH2)

[0067] KCNH2 is a key gene encoding the α subunit of the human fast-activated delayed rectifier potassium current (IKr) channel, also known as hERG (human Ether-à-go-go-Related Gene). The protein expressed by this gene is mainly distributed on the cardiomyocyte membrane and participates in the cardiac action potential repolarization process by regulating potassium ion efflux, which is crucial for maintaining normal heart rhythm. Mutations in the KCNH2 gene can lead to hERG channel dysfunction, causing type 2 long QT syndrome, characterized by delayed ventricular repolarization, QT interval prolongation, and an increased risk of torsades de pointes and sudden cardiac death. Furthermore, the hERG channel is easily non-specifically blocked by various drugs (such as antiarrhythmics, antibiotics, and antidepressants), making it an important target for cardiac safety assessment in drug development. Recent studies have also found potential links between KCNH2 and epilepsy, mental illness, and cancer.

[0068] QT syndrome (also known as long QT syndrome)

[0069] QT syndrome is a cardiac electrophysiological abnormality characterized by a prolonged QT interval on an electrocardiogram (ECG), significantly increasing the risk of ventricular arrhythmias (such as torsades de pointes), syncope, and sudden cardiac death. It can be classified into congenital and acquired types based on etiology. Clinical manifestations are diverse; mild cases may be asymptomatic, while severe cases may result in sudden death, and cardiac arrest can even occur in infancy. Treatment primarily involves beta-blockers (such as metoprolol), which can shorten the QT interval and reduce the risk of sudden death. High-risk patients may require implantation of an intracardiac defibrillator (ICD) or left cervical sympathectomy. Furthermore, avoiding triggering factors (such as certain medications or strenuous exercise) and regular ECG monitoring are crucial for management.

[0070] Cystic fibrosis transmembrane conductance modulator (CFTR)

[0071] CFTR is an important ion channel protein located on the cell membrane, primarily responsible for regulating the transmembrane transport of chloride and bicarbonate ions, which is crucial for maintaining fluid and electrolyte balance. It belongs to the ATP-binding cassette (ABC) transporter superfamily, but its function is more inclined to regulate the opening and closing of ion channels through ATP hydrolysis rather than direct energy consumption for active transport. The CFTR protein structure includes two transmembrane domains (forming channel pores), two nucleotide-binding domains (binding to ATP and regulating channel opening and closing), and a unique regulatory domain (activating channel function through phosphorylation). Mutations in the CFTR gene are the root cause of cystic fibrosis (CF), a common and fatal genetic disease. The most common mutation type, ΔF508, leads to abnormal protein folding, preventing normal transport to the cell membrane, resulting in abnormal mucus secretion and organ dysfunction (such as lung infections, pancreatitis, and infertility). In recent years, targeted drugs for CFTR dysfunction (such as the potentiator Ivacaftor and the corrector Lumacaftor) have significantly improved patient prognosis by repairing the folding of the mutant protein or enhancing its activity.

[0072] Cystic fibrosis (CF)

[0073] Cystic fibrosis is an autosomal recessive genetic disorder caused by mutations in the CFTR gene, primarily affecting the respiratory tract, digestive system, and sweat glands. Patients with CFTR protein dysfunction experience abnormal chloride ion transport, resulting in abnormally thick mucus secretions that can obstruct the bronchi and pancreatic ducts. Typical symptoms include persistent cough, recurrent lung infections, dyspnea, malabsorption, growth retardation, and neonatal meconium ileus.

[0074] Phosphoribose pyrophosphate aminoacyltransferase (PPAT)

[0075] 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).

[0076] Cytidine triphosphate synthase 1 (CTPS1) / Cytidine triphosphate synthase 2 (CTPS2)

[0077] 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.

[0078] Glutamine-6-phosphofructoaminotransferase 1 (GFPT1) / Glutamine-6-phosphofructoaminotransferase 2 (GFPT2)

[0079] 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.

[0080] Glutamine-dependent NAD + Synthesizer (NADSYN1)

[0081] Nicotinamide adenine dinucleotide synthase 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.

[0082] GMP synthase (GMPS)

[0083] 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.

[0084] Asparaginase (ASRGL1)

[0085] 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.

[0086] Pharmaceutical Composition

[0087] The present invention also provides a pharmaceutical composition comprising:

[0088] 1) A deaminase or transaminase as the first active ingredient, an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase;

[0089] 2) A cofactor or metabolic substrate of the first active ingredient as a second active ingredient; and

[0090] 3) Pharmaceutically acceptable carrier.

[0091] In one specific embodiment, the first active ingredient is asparagine synthase, and the second active ingredient is glutamine.

[0092] The pharmaceutical composition can be used for:

[0093] (1) Deamination of aspartic acid residues in KRAS G12D protein to generate KRAS G12N, i.e., aspartic acid residues; prevention and / or treatment of pancreatic cancer, colorectal cancer, or non-small cell lung cancer; improvement of the sensitivity of the above tumors to immunotherapy (such as T-cell vaccines, tumor vaccines, immune checkpoint inhibitors); improvement of the sensitivity of the above tumors to traditional treatments (such as targeted therapy, chemotherapy, radiotherapy).

[0094] (2) Deamination of aspartic acid residues in KRAS G13D protein to generate KRAS G13N, i.e., aspartic acid residues; prevention and / or treatment of colorectal cancer or lung cancer; improvement of the sensitivity of the above tumors to immunotherapy (such as T-cell vaccines, tumor vaccines, immune checkpoint inhibitors); improvement of the sensitivity of the above tumors to traditional treatments (such as targeted therapy, chemotherapy, radiotherapy).

[0095] (3) Deamination of aspartic acid residues in BRAF V600D protein to generate BRAF V600N, i.e., aspartic acid residues; prevention and / or treatment of melanoma or colorectal cancer; improvement of the sensitivity of the above tumors to immunotherapy (such as T-cell vaccines, tumor vaccines, immune checkpoint inhibitors); improvement of the sensitivity of the above tumors to traditional treatments (such as targeted therapy, chemotherapy, radiotherapy).

[0096] (4) Deamination of aspartic acid residues in CFTR G551D protein to generate CFTR G551N, i.e., aspartic acid residues; prevention and / or treatment of cystic fibrosis; and enhancement of the sensitivity of cystic fibrosis to Ivacaftor (CFTR enhancer).

[0097] (5) Deamination of aspartic acid residues in KCNH2 N588D protein to generate KCNH2N588N, i.e., aspartic acid residues; prevention and / or treatment of hereditary arrhythmias (such as long QT syndrome); and improvement of the sensitivity of hereditary arrhythmias to β-blockers.

[0098] (6) Deamination of mutated aspartic acid residues in GBA proteins; prevention and / or treatment of lysosomal storage diseases (such as Gaucher disease); and enhancement of the sensitivity of lysosomal storage disorders to enzyme replacement therapy, substrate reduction therapy, or gene therapy.

[0099] (7) Deamination of mutated aspartic acid residues in β-globin; prevention and / or treatment of sickle cell anemia; and enhancement of the sensitivity of sickle cell anemia to hydroxyurea or stem cell transplantation.

[0100] The pharmaceutical compositions of the present invention comprise a combination of active ingredients within a safe and effective range, and a pharmaceutically acceptable carrier.

[0101] "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.

[0102] 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.

[0103] 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.

[0104] When using pharmaceutical preparations, safe and effective amounts of active ingredients are combined and administered to mammals.

[0105] 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.

[0106] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] Compared with the prior art, the main advantages of the present invention include:

[0111] 1. This invention discovers that the asparagine synthase ASNS deaminates protein aspartic acid residues (such as the KRAS G12D mutation), effectively reducing the expression level of mutant aspartic acid residues, thereby inhibiting downstream pathways (such as the ERK / AKT pathway) and treating diseases carrying aspartic acid residue mutations.

[0112] 2. The active ingredient ASNS used in this invention is delivered to the relevant concurrent areas using liposome encapsulation and other technologies, which is safe and efficient; another active ingredient ASNS used in this invention has a cofactor glutamine, which is a naturally occurring metabolite in the human body and has high safety for the human body.

[0113] 3. This invention is applicable to a variety of diseases carrying aspartic acid residue mutations, such as pancreatic cancer, colorectal cancer, non-small cell lung cancer, melanoma, cystic fibrosis, and hereditary arrhythmia, and can achieve the goal of "treating different diseases with the same method".

[0114] 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.

[0115] Example 1. ASNS and glutamine inhibit G12D expression in pancreatic cancer cells.

[0116] In the pancreatic cancer cell line Aspc-1 carrying KRAS G12D, the cells were transiently transfected with an ASNS plasmid overexpressing the Flag tag or its empty vector control. After 4 hours of glutamine-free culture, 2 mM glutamine was added. Cells were collected after 12 hours, and intracellular proteins were isolated for immunoblotting analysis.

[0117] The results are as follows Figure 1 As shown, KRAS G12D-mutant pancreatic cancer cells Aspc-1, after overexpression of ASNS or treatment with glutamine, significantly inhibited the protein expression level of KRAS G12D in cells and suppressed the expression of phosphorylated ERK, suggesting that ASNS or its metabolic substrates may have a deamination effect.

[0118] Example 2. Asparaginase ASNS catalyzes the deamination of KRAS G12D in vitro.

[0119] Purified Flag-tagged ASNS and His-tagged KRAS G12D protein, along with glutamine at concentrations of 0, 0.1, 0.5, and 1 mM, were reacted in a buffer containing MgCl2 and ATP at 30°C for 2 h. Samples were then analyzed by Western blotting using the corresponding antibodies.

[0120] The results are as follows Figure 2 As shown, in the presence of glutamine, asparaginase ASNS can reduce the expression of KRAS G12D and promote the expression of KRAS G12N in vitro; and as the concentration of glutamine increases, the expression of KRAS G12D gradually decreases, while the expression of KRAS G12N gradually increases.

[0121] Furthermore, mass spectrometry detected the generation of the KRAS G12N peptide (LVVVGANGVGK) in the above reaction system, indicating that ASNS can deaminate and convert KRAS G12D protein into KRASD12N in vitro.

[0122] Example 3. Glutamine promotes KRASD12N expression and inhibits ERK phosphorylation in vivo.

[0123] In the KRAS G12D stable mutant cell lines PANC1 and AsPC-1, the samples were first treated with glutamine-free conditioned medium for 4 h, and then treated with 0, 0.1, 0.5, and 2 mM glutamine for 12 h. The samples were then detected by Western blotting using the corresponding antibodies.

[0124] The results are as follows Figure 3 As shown, in the PANC1 and AsPC-1 cell lines, glutamine can promote the expression of KRAS D12N and inhibit the phosphorylation of downstream ERK of KRAS G12D in a dose-dependent manner, while glutamine addition promotes ASNS expression. This suggests that glutamine can promote the deamination of KRAS G12D to form KRASD12N catalyzed by ASNS in vivo.

[0125] Example 4. ASNS K378 and KRASD12 are important binding sites.

[0126] Molecular docking simulations of the KRAS G12D protein (PDB: 7RPZ) and ASNS protein (PDB: 8SUE) from PDB data showed that ANSI473 interacts with KRAS R102, ANST474 with KRAS H95, ANSY375 with KRAS Y32, and ANSK378 and ANSK539 with KRASD12 to form hydrogen bonds.

[0127] KRASD12A, KRAS Y32A, KRAS H95A, and KRAS R102A mutants tagged with Flag were constructed and transfected into HEK-293T cells for IP detection. Western blotting experiments showed that... Figure 4 Compared with the Flag-KRAS WT group, the Flag-KRASD12A mutant group significantly reduced the binding to ASNS, suggesting that KRASD12 and ASNS K378 are important binding sites for both.

[0128] Example 5. Glutamine supplementation can inhibit the growth of AsPC-1 tumors in nude mice.

[0129] Five 10⁶ KRAS G12D stably mutant AsPC-1 cells were transplanted subepithelially into 6-week-old nude mice of the BALB / c-Nude strain. After tumor formation, mice were administered glutamine daily by gavage at doses of 500 mg / kg, 2000 mg / kg, and 8000 mg / kg, with pure water as the control group (NC).

[0130] The results are as follows Figure 5 As shown, after collecting mouse tumors on day 21, administration of glutamine at doses of 2000 mg / kg and 8000 mg / kg significantly inhibited tumor volume and weight in mice. This indicates that glutamine supplementation can inhibit the growth of AsPC-1 xenografts in vivo.

[0131] Example 6. Asparaginase ASNS catalyzes the deamination of aspartic acid-containing mutant peptides in vitro.

[0132] The purified Flag-tagged ASNS was reacted with an aspartate-mutant peptide and 1 mM glutamine in a buffer containing MgCl2 and ATP at 30 °C for 2 h. The deamination peptide sequence was detected by liquid chromatography-mass spectrometry, and the proportions were calculated. The results showed that in the presence of glutamine, asparaginase ASNS can catalyze the deamination reaction of the peptide in vitro.

[0133] Table 1. Results of in vitro deamination of aspartic acid-containing mutant peptides catalyzed by asparaginase ASNS.

[0134]

[0135] 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. Use of a deaminase or transaminase and optionally a cofactor of said deaminase or transaminase, characterized in that, For the preparation of a drug / formulation, said drug / formulation having uses selected from the group consisting of: a. Directly deaminate the aspartic acid residue of a protein or polypeptide carrying a mutation, thereby converting the aspartic acid at the mutation site into asparagine; b. Catalyzing the deamination of proteins or peptides carrying mutations in aspartic acid residues in vitro; c. Inhibit tumor growth; and / or d. To treat diseases carrying mutations in aspartic acid residues or to improve the sensitivity of said diseases to immunotherapy or conventional treatment.

2. The use as described in claim 1, characterized in that, The deaminase or transaminase is asparagine synthase (ASNS); the cofactor is glutamine.

3. The use as described in claim 1, characterized in that, The proteins carrying mutations in aspartic acid residues include, but are not limited to, the following: Kirsten rat sarcoma virus oncogene homolog (KRAS), Harvey rat sarcoma virus oncogene homolog (HRAS), neuroblastoma Ras virus oncogene homolog (NRAS), B-Raf proto-oncogene serine / threonine protein kinase (BRAF), cystic fibrosis transmembrane conductance regulator (CFTR) protein, potassium voltage-gated channel subfamily H member 2 (KCNH2), glucocerebroside lipase (GBA), β-globin, phenylalanine hydroxylase (PAH), fibrin-1 (FBN1), dystrophin (DMD), type I collagen α1 chain (COL1A1), or combinations thereof.

4. The use as described in claim 1, characterized in that, The transaminases include, but are not limited to, the following group: 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), or protein-glutamine gamma-glutamyltransferase 2 (TGM2).

5. The use as described in claim 1, characterized in that, The deaminases include, but are not limited to, the following group: 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), HSV UL37, HSVORF75 / ORF75c, E. coli CNFs, E. coli Cycle-inhibiting factors (Cifs), Pasteurella multocida toxin (PMT), Vibrio parahaemolyticus VopC, Burkholderia pseudomallei Burkholderia lethal factor 1 (BLF1), or Shigella flexneri OspI.

6. The use as described in claim 1, wherein the cofactor includes, but is not limited to, the following group: glutamine, aspartic acid, 5-phospho-alpha-D-ribose 1-diphosphate, D-fructose 6-phosphate, and deamido-NAD. + , or a combination thereof.

7. The use as described in claim 1, characterized in that, The diseases carrying aspartate residue mutations include, but are not limited to, the following groups: pancreatic cancer, pancreatic ductal carcinoma, colorectal cancer, non-small cell lung cancer, lung cancer, melanoma, papillary thyroid carcinoma, cystic fibrosis, hereditary arrhythmias (such as long QT syndrome), lysosomal storage diseases (such as Gaucher disease), sickle cell anemia, phenylketonuria, Marfan syndrome, dystrophinosis, osteogenesis imperfecta, or combinations thereof.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) The first active ingredient is the deaminase or transaminase as described in claim 1, an mRNA / nucleic acid construct expressing the deaminase or transaminase, or a vector expressing the deaminase or transaminase; Optional (ii) a second active ingredient, wherein the second active ingredient is a cofactor of the said deaminase or transaminase; and (iii) Pharmaceutically acceptable carriers.

9. A reagent kit, characterized in that, The kit includes: A first container and a first reagent within the container, wherein the first reagent is a deaminase or transaminase as described in claim 1, an mRNA / nucleic acid construct expressing the deaminase or transaminase, or a vector expressing the deaminase or transaminase; and An optional second container and a second reagent within the container, wherein the second reagent is a cofactor of the deaminase or transaminase.

10. A method for deamination of a protein or polypeptide containing a mutation of aspartic acid residue in vitro, characterized in that, The method includes the following steps: A protein or polypeptide containing an aspartic residue mutation is co-incubated with a reaction system containing the deaminase or transaminase as described in claim 1, or an mRNA / nucleic acid construct for expressing the deaminase or transaminase, or a vector for expressing the deaminase or transaminase, thereby obtaining a protein or polypeptide with an aspartic residue mutation to asparagine.

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