Histamine H3 receptor protein truncated body with bias, gene and application
By constructing and expressing the biased histamine H3 receptor protein truncator H3R339, the problem of difficult to distinguish and regulate the H3 receptor signaling pathway in the prior art is solved, and the clear regulation of histamine H3 receptor signaling is achieved. Provides the potential for drug development and gene therapy with high efficacy and low toxic side effects.
Patent Information
- Application Number
- CN202411273895.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art is difficult to distinguish and regulate the different signaling pathways of histamine H3 receptors, which makes it difficult to isolate the efficacy and side effects of drugs. In gene editing technology, it lacks the development of efficient and low-toxic drugs for H3 receptors.
The biased histamine H3 receptor protein truncator H3R339 was constructed by artificially modifying the protein, and the protein was expressed in cells using gene editing technology. The bias of its downstream signal was verified through signal transduction experiments, providing the development of targeted H3 receptors Theoretical basis and model of body medicine.
The clear bias regulation of downstream signals of histamine H3 receptors has been achieved, providing the potential to develop drugs with high efficacy and low toxic side effects, and providing new application pathways for gene therapy.
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Figure CN119161450A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a biased histamine H3 receptor protein truncation, a gene and an application thereof. Background Art
[0002] Gene editing technology, especially CRISPR-Cas9 technology, is used to treat certain hereditary recessive diseases, which cause abnormal red blood cell morphology and sickle shape. Patients may experience symptoms such as anemia, severe pain, immunodeficiency, and multiple organ failure. Recently, CRISPR-Cas9 gene editing technology has made a breakthrough in the treatment of sickle cell disease. In 2023, Dr. A. Sharma's team from the Department of Bone Marrow Transplantation and Cell Therapy at St. Jude Children's Research Hospital in the United States led a study published in the New England Journal of Medicine. They used CRISPR-Cas9 technology to edit the HBG1 and HBG2 promoters to treat sickle cell disease. Clinical trials have shown (NCT04443907) that the autologous OTQ923 drug has shown safety and effectiveness in treating patients with severe sickle cell disease.
[0003] Histamine H3 receptors (H3R) are widely distributed in the central nervous system and have been shown to be involved in neurobehavioral disorders such as Alzheimer's disease, attention deficit hyperactivity disorder, epilepsy, stress, anxiety, Parkinson's disease, etc. In addition, it has been found that H3R agonists and antagonists may have therapeutic effects on diseases such as asthma, hypertension, septic shock, heart failure, acute myocardial infarction, sleep disorders, obesity and pain.
[0004] Recent studies have shown that H3R antagonists reduce neuronal apoptosis and cerebral infarction volume in the acute phase after cerebral ischemia, but their effects are unrelated to histaminergic neurons. At the same time, H3R antagonists can promote angiogenesis after focal cerebral ischemia, which is unrelated to histaminergic neurons but is related to H3R in vascular endothelial cells, involving the regulation of Annexin A2 (ANXA2). Therefore, H3R has the potential to become a new target for the treatment of ischemic stroke. Since H3R is mainly distributed in the central nervous system and related drugs have few peripheral side effects, histamine H3 receptors are targets with great clinical application prospects.
[0005] H3R can mediate downstream Gi protein and repressor protein signaling pathways respectively. Current studies have shown that different signaling pathways can be involved in different physiological functions. For example, the repressor protein pathway of NTSR1 mediates the therapeutic effect of reducing addiction, while its G protein pathway mediates the side effects of hypothermia, hypotension and movement disorders. Therefore, the development of a biased H3R receptor will be conducive to further distinguishing the functions of different H3R signaling pathways and will be conducive to the development of drugs with high efficacy and low toxic side effects. At the same time, biased receptors also have the potential for clinical application through gene editing technology and have extremely high gene therapy potential.
[0006] If researchers artificially modify H3R through molecular biological methods and develop a biased exogenous protein, it will have extremely important medicinal value. Summary of the invention
[0007] Based on this, the present invention provides an artificially modified exogenous protein with bias. An expression vector containing the exogenous protein gene is obtained by in vitro amplification, and the protein is expressed in tumor cells by liposome transfection, so that the downstream signal of the exogenous protein is proved to be biased through signal transduction experiments.
[0008] Specifically, the present invention provides a truncated histamine H3 receptor protein with bias, and the amino acid sequence of the truncated histamine H3 receptor protein is shown in SEQ ID NO.1.
[0009] The present invention also provides a gene encoding the truncated version of the histamine H3 receptor protein.
[0010] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.
[0011] The invention also provides an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.
[0012] The present invention also provides an expression vector, the expression vector comprising the gene. In the present embodiment, plasmid pcDNA3.1 is selected as an example.
[0013] The present invention also provides the use of the histamine H3 receptor protein truncation, or the gene, or the expression box, recombinant vector, recombinant microorganism or transgenic cell line containing the gene in the preparation of a biased H3 receptor drug.
[0014] When used, the gene is introduced into cells to express the histamine H3 receptor protein truncations.
[0015] The present invention also provides a biased H3 receptor drug, the active ingredient of which includes the histamine H3 receptor protein truncation, or the gene, or an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.
[0016] The present invention utilizes the independent signal pathway downstream of the H3 receptor protein and constructs a variant H3 receptor H3R339 with G protein bias through artificial modification. Signal transduction experiments show that the modified protein exhibits good G protein bias, can be used to study the mechanism of histamine H3 receptor biased signal transduction, and provides a theoretical basis and model for the development of drugs targeting H3 receptors. It has certain application prospects in the field of medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the plasmid map of pcDNA3.1-H3R-WT, on which a biased receptor was obtained through modification; and it serves as a positive control for G protein activation in response to histamine.
[0018] Figure 2 The plasmid map is pcDNA3.1-H3R-WT-RLUC8, which serves as a positive control for the recruitment of the repressor protein in response to histamine.
[0019] Figure 3 The plasmid map of pcDNA3.1-H3R339-RLUC8 is used to detect the recruitment of repressor proteins in response to histamine.
[0020] Figure 4 The plasmid map of pcDNA3.1-H3R339 is used to detect G protein activation in response to histamine.
[0021] Figure 5 The curve of H3R339 recruiting repressor proteins. BRET assay detected 48 hours after transfection and found that H3R339 could not recruit repressor proteins.
[0022] Figure 6 The graphs are respectively H3R339 activation G protein curves. NanoBit assay was performed 48 hours after transfection, and the maximum effect of H3R339 activation of G protein was calculated to be 101.63% of WT. This indicates that H3R339 mutation has no significant effect on the downstream G protein signal of H3R. DETAILED DESCRIPTION
[0023] Example 1: Cloning and construction of H3 receptor protein expression vector pcDNA3.1-H3R-WT
[0024] Gene synthesis was performed at GENEWISE according to the complete gene sequence of the H3 receptor protein (ENSG00000101180). The nucleotide sequence of the gene is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8. The H3 receptor sequence was constructed into the pcDNA3.1 vector by homologous recombination. The sequences of the primers used in the construction are shown in Table 1.
[0025] Table 1 Primers
[0026]
[0027]
[0028] The pcDNA3.1-H3R-WT plasmid DNA fragment was obtained by PCR method. The PCR reaction system is shown in Table 2.
[0029] Table 2 PCR amplification system
[0030] Template DNA 1μl Forward primer (10 μM) 1.5μl Reverse primer (10 μM) 1.5μl High-fidelity Taq enzyme 15μl Double distilled water 10μl DMSO 1μl Total volume 30μl
[0031] PCR reaction conditions: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, for a total of 18 cycles; extension at 72°C for 10 min.
[0032] The obtained DNA fragment was transformed into competent cells DH5α for amplification, and positive colonies were extracted and sequenced for verification. The eukaryotic expression vector pcDNA3.1-H3R-WT was obtained, and its map is shown in Figure 1 shown.
[0033] Example 2: Cloning and construction of H3 receptor protein from expression vector pcDNA3.1-H3R339
[0034] Deletion mutation primers were designed based on pcDNA3.1-H3R-WT to construct a receptor truncation in which 235-342 bp was replaced with GGAGGAGGAG and 429-445 bp was truncated. The sequences of the mutation primers are shown in Table 3.
[0035] Table 3 Mutation primers
[0036]
[0037] The pcDNA3.1-H3R-WT plasmid prepared in Example 1 was used as a template to obtain the pcDNA3.1-H3R339 plasmid DNA fragment by mutation PCR method. The PCR reaction system and PCR reaction conditions were the same as those in Example 1.
[0038] The truncated form obtained by mutation of the H3 receptor is named as H3R339 protein. The amino acid sequence of the H3R339 protein is shown in SEQ ID No.1; the gene sequence of H3R339 is shown in SEQ ID No.2.
[0039] The obtained pcDNA3.1-H3R339 plasmid DNA fragment was transformed into competent cells DH5α for amplification, and positive colonies were extracted and sequenced for verification. Figure 4 shown).
[0040] Example 3: Biased effects of H3R339 on downstream signals
[0041] (1) H3R339 significantly affects the recruitment of repressor proteins
[0042] Histamine family receptors, as G protein-coupled membrane receptors, can be phosphorylated by GRKs, thereby recruiting repressor proteins. In the BRET experiment, Rluc (sequence as shown in SEQ ID NO.9) and Venus (sequence as shown in SEQ ID NO.10) sequences are respectively carried on H3R and repressor proteins. When these two recombinant proteins interact with each other, the energy emitted by luciferase can be transferred to the fluorescent protein, thereby detecting a yellow light peak in the luminescence spectrum. The BRET experiment was used to determine the ability of H3R to recruit repressor proteins.
[0043] In the BRET experiment, the plasmid containing H3R-Rluc (pcDNA3.1-H3R-WT-RLUC8) was transfected into Figure 2 The plasmid map of pcDNA3.1-H3R339-RLUC8 is shown in Figure 3 The plasmids of 1:4 were transfected into HEK293T cells, and about 24 hours later, they were plated into white opaque 96-well plates coated with adhesion factors. After 24 hours, the cells were rinsed twice with PBS, and then 45 microliters of buffer (HBSS, 5mM HEPES pH 7.4 and 0.1% BSA) were added and incubated for 30 minutes. 10μM coelenterazine 400a was added as a fluorescent substrate, incubated at room temperature for 5 minutes, and fluorescence was detected by a multifunctional microplate reader, with an excitation light of 410nm and an emission light of 515nm; after adding histamine with a gradient dilution, 15 cycles were measured again.
[0044] Figure 5 The curve of H3R339 recruiting repressor proteins. BRET assay detected 48 hours after transfection and found that H3R339 could not recruit repressor proteins.
[0045] (2) H3R339 does not affect G protein activation
[0046] Histamine family receptors, as G protein-coupled membrane receptors, can activate G proteins, causing the Gα, Gβ and Gγ subunits of G proteins to dissociate and activate downstream signaling molecules. In the NanoBit experiment, SmBit (sequence shown in SEQ ID NO.12) and LgBit sequence (sequence shown in SEQ ID NO.13) are respectively attached to Gα and Gγ. When the spatial distance between the two is close enough, they will have hydrolase activity and hydrolyze the fluorescent substrate to emit fluorescence. The NanoBit experiment is used to measure the ability of H3R to dissociate Gi protein.
[0047] In the NanoBit experiment, we used LIPO transfection reagent to transfect plasmids containing H3R-WT / H3R339, Gαi-SmBit (sequence as shown in SEQ ID NO.14), Gβ (sequence as shown in SEQ ID NO.15), and Gγ-LgBit (sequence as shown in SEQ ID NO.16) gene fragments into HEK293T cells at a ratio of 6:2:1:1. After about 24 hours, the cells were plated in a white-bottomed opaque 96-well plate coated with adhesion factors. After 24 hours, the cells were rinsed twice with PBS, and then 10μM coelenterazine h was added as a fluorescent substrate (the buffer was HBSS, 5mM HEPES pH7.4 and 0.1% BSA were added). Incubated at room temperature for 1 hour, luminescence was measured with a multifunctional microplate reader; after adding gradient dilutions of histamine, 15 cycles were measured again.
[0048] Figure 6 The graphs are respectively H3R339 activation G protein curves. NanoBit assay was performed 48 hours after transfection, and the maximum effect of H3R339 activation of G protein was calculated to be 10 1.63% of WT. This indicates that the H3R339 mutation has no significant effect on the downstream G protein signal of H3R.
[0049] In summary, the H3R receptor protein truncation we constructed: H3R339 protein, has a G protein-biased downstream signal.
Claims
1. A truncated histamine H3 receptor protein with bias, characterized in that: The amino acid sequence of the histamine H3 receptor protein truncation is shown in SEQ ID NO.
1.
2. A gene encoding the histamine H3 receptor protein truncated form according to claim 1.
3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the gene according to claim 2 or 3.
5. An expression vector, characterized in that: The expression vector comprises the gene according to claim 2 or 3.
6. Use of the histamine H3 receptor protein truncate according to claim 1, or the gene according to claim 2 or 3, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene according to claim 2 or 3 in the preparation of a biased H3 receptor drug.
7. The use according to claim 6, characterized in that When used, the gene is introduced into cells to express the histamine H3 receptor protein truncations.
8. A H3 receptor-biased drug, characterized in that: The active ingredient of the biased H3 receptor drug includes the histamine H3 receptor protein truncation according to claim 1, or the gene according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the gene according to claim 2 or 3.