CN114480291A - Cell lines and methods for detecting biological activity of dopamine D4 target compound - Google Patents

Abstract

The invention discloses a cell line and a method for detecting the biological activity of a dopamine D4 target compound, wherein the cell line comprises a polynucleotide for coding a dopamine D4 receptor protein and a polynucleotide for coding luciferase with the expression controlled by a CRE transcription regulatory element. The invention also discloses a method for detecting the biological activity of the dopamine D4 target compound by using the cell line. The cell line can rapidly, accurately and efficiently detect the biological activity of the dopamine D4 target therapeutic drug. In addition, the detection method provided by the invention can completely meet the requirement of detecting the biological activity of the small-molecule compound aiming at the dopamine D4 receptor without complex instruments or operation steps.

Description

Cell lines and methods for detecting biological activity of dopamine D4 target compound

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a cell line for detecting biological activity of a dopamine D4 target compound, which comprises a polynucleotide for coding a dopamine D4 receptor protein and a polynucleotide for coding luciferase expressed under the control of a CRE transcription regulatory element; the invention also relates to a detection method for detecting the biological activity of the dopamine D4 target compound by using the cell line.

Background

The dopaminergic system plays important roles in neuromodulation, such as motor control, motivation, reward, cognitive function, maternal and reproductive behavior. Dopamine (Dopamine) is a neurotransmitter that is produced at the neuronal terminals by the sequential hydroxylation and decarboxylation of tyrosine and loaded into synaptic vesicles by the vesicular monoamine transporter 2 for exocytosis release. The released dopamine can be reuptake by dopamine transporters and can also be degraded by catechol-O-methyltransferase and presynaptic monoamine oxidase. Dopamine acts by binding to dopamine receptors located on the membrane of the target cell.

Dopamine receptors belong to the GPCRs superfamily, mainly including five types D1, D2, D3, D4 and D5. Among them, D1 and D2 are dopamine receptors most expressed in the brain, and they are hardly simultaneously expressed in the same cell. According to the structural and pharmacological properties of dopamine receptors, it can be divided into two main classes: receptors of class D1, including D1 and D5; receptors of class D2, including D2, D3 and D4. All dopamine receptors are metabotropic receptors, of which the D1 and D5 receptors are associated with G_αs/olfProtein coupling, stimulating the production of the second messenger cAMP; d2, D3 and D4 receptors with G_αi/oProtein coupling, inhibiting intracellular cAMP production.

The D1 receptor is expressed primarily in the caudate nucleus (CPu), nucleus accumbens (Acb), Optic Tract (OT), cortex (Cx), and amygdala. The D5 receptor is restricted in expression compared to the D1 receptor and is only expressed in the hippocampus, lateral papillary nuclei and subthalamic nucleus. The D2 receptor is mainly expressed in the brain in CPu, OT and Acb, as well as in the substantia nigra and Ventral Tegmental Area (VTA), which emit dopamine fibers, suggesting presynaptic localization of the D2 receptor. In contrast, receptors of class D1 have only extensive postsynaptic localization. Outside the brain, the D2 receptor is also localized to the retina, kidney, vasculature and pituitary. The distribution of the D3 receptor is limited to only the islands of caliija, the septal nucleus, the hypothalamus and thalamus, and certain regions of the cerebellum. The D3 receptor is also distributed in the substantia nigra pars compacta, suggesting that it also has presynaptic localisation. High expression of the mRNA of the D4 receptor is seen in the prefrontal cortex, amygdala, olfactory bulb, hippocampus, thalamus and midbrain.

Currently, the signal transduction pathways for dopamine receptor activation mainly include: the activation of adenylate cyclase, or the inhibition of calcium signaling. The D1 class of receptors positively regulate cyclic adenosine monophosphate (cAMP) levels. Further activates Protein Kinase A (PKA), which phosphorylates cytoplasmic and nuclear factors, regulates cellular metabolism including ion channel function, desensitizes transmembrane G protein-coupled receptors, and leads to the release of cellular neurotransmitters. The D2 class of receptors generally inhibit Adenylate Cyclase (AC) activity. The D2 class of receptors were first found to inhibit cAMP levels in pituitary and striatal cells. The D2 receptor inhibits AC by coupling to a signaling pathway and can be blocked by Pertussis Toxin (PTX). The D3 receptor inhibited endogenous cAMP levels in several cell lines, but to a lesser extent than the D2 receptor. The D4 receptor has been reported to inhibit the aggregation of cAMP in the retina and some cell lines.

Numerous studies have shown that dopamine receptor D4 (subtype D4) is associated with Attention Deficit Hyperactivity Disorder (ADHD), cancer metastasis, and even erectile dysfunction. Similar dopamine receptor subtypes play a crucial role in diseases such as schizophrenia, addiction, alzheimer's disease, depression and parkinson's disease.

The D4 receptor signaling pathway is difficult to detect compared to the D1, D5 receptors, the D1 receptor being a Gs-coupled GPCR, such receptors including, for example, DRD1(Homo sapiens (human)), Dop1R1(Drosophila melanogaster (free flow), BmDopR1 (silolworm), and BmDopR2 (silolworm), etc., when these D1 receptors are activated by ligands, their signals are rapidly transmitted downstream, and the intrinsic activity of the ligands (e.g., agonists, partial agonists, and antagonists) can be readily reflected by detecting changes in intracellular second messenger cAMP, unlike the reports on the D4 receptor, which show that, unlike other Gi 4 receptor overexpressing cell lines, the downstream coupled signaling pathway changes are very difficult to detect (e.g., second messenger changes in GPCRs), and this difference is not clear at present time for the reasons why the GPCRA 7-300,564: PET-800,564,564,300,361 (Japanese laid open-European Pharma,564,361), although some D4 receptor agonists reflect the effect of the compound by causing cell division, resulting in a change in the number of cells, this approach is not very high in throughput, not sufficiently sensitive and time consuming and has not been widely used. In summary, there are only reports about the D4 target drug activity detection method, so that the differentiation of specific functions of D4 and other dopamine receptors is influenced, and the development of D4 target drugs is hindered, so that D4 target drug detection is still an urgent problem to be solved in new drug development.

Disclosure of Invention

In order to overcome the defect that a method for detecting the biological activity of a dopamine D4 target drug is lacked in the prior art, the invention provides a cell line and a detection method for detecting targeted dopamine D4 by using the cell line.

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The words "preferred", "preferably", "more preferably", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention. The sources of components not mentioned in the present invention are all commercially available.

Before the present proteins, nucleotide sequences, and methods are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of protein chemistry and biochemistry, molecular biology, microbiology and recombinant DNA technology, which are within the skill of the art.

After earnest research for solving the problems, the inventor provides a novel cell line which is CHO-K1 cells capable of stably expressing dopamine D4 receptor and luciferase expressed under the control of CRE transcription regulatory element, and the cell line can be used as a cell model for accurately and efficiently detecting the biological activity of a dopamine D4 target compound.

In addition, the inventor successfully develops a method for detecting the biological activity of the dopamine D4 target compound based on the cell line, and the method comprises the steps of contacting the cell line with a candidate drug, measuring the change of luciferase level expressed by cells, calculating relative chemiluminescence units of the candidate drug, and obtaining a dose-response curve of the candidate drug to calculate the biological activity data of the candidate drug.

The principle of the detection method is as follows: by contacting the cell line (i.e., a cell line of the invention comprising (a) a polynucleotide encoding dopamine D4 receptor protein and (b) a polynucleotide encoding luciferase expressed under the control of CRE transcription regulatory elements) with a candidate drug, provided that the candidate drug is a compound having dopamine D4 receptor agonistic activity, binding to the dopamine D4 receptor protein expressed by polynucleotide (a) inhibits intracellular adenylate cyclase activity, resulting in a decrease in intracellular cAMP levels and thus a decrease in Protein Kinase A (PKA) phosphorylation levels. When the phosphorylation level of PKA is reduced, the level of phosphorylated CREB bound to CRE transcriptional regulatory element having promoter function in polynucleotide (b) is correspondingly reduced, inhibiting the expression of luciferase gene downstream thereof, thereby reducing the level of fluorescent signal in the final detection system. Therefore, the biological activity of the candidate drug is finally calculated by the decrease in the level of the fluorescence signal exhibited.

Cell lines

In a first aspect, the present invention provides a cell line for detecting the biological activity of a dopamine D4 target compound, comprising (a) a polynucleotide encoding a dopamine D4 receptor protein; and (b) a polynucleotide encoding a luciferase whose expression is controlled by a CRE transcription regulatory element.

The term "polynucleotide" refers to DNA or RNA. Wherein a polynucleotide (nucleic acid, gene) is a polynucleotide (nucleic acid, gene) that "encodes" a desired protein. The term "encode" as used herein means that a desired protein is expressed in a state where it has its activity; in addition, "encoding" includes both the meaning of encoding a desired protein with a linked structural sequence (exon) or by a mediating sequence (intron).

The polynucleotide (a) of the present invention is contained in a cell in a state that can be used for expressing the dopamine D4 receptor protein, and specifically means that the polynucleotide (a) is placed in a suitable transcription regulatory region of the cell, particularly under the control of a suitable promoter, which is not particularly limited in the present invention as long as it has promoter activity in the cell containing the polynucleotides (a) and (b). Any known promoter may be used, for example, in mammalian cells, viral promoters such as CMV promoter, SV40 promoter, EF-1. alpha. promoter, UbC promoter, etc. may be used.

The dopamine D4 receptor protein encoded by polynucleotide (a) can be derived from various organisms, and can be mammalian or non-mammalian, preferably mammalian, e.g., rat, mouse, zebrafish, primate, etc.; among them, human or rodent cells are more preferable.

For the polynucleotide sequence of the present invention (a) encoding dopamine D4 receptor protein, wherein the dopamine D4 receptor protein may be a protein having a modified amino acid sequence or a protein having an unmodified amino acid sequence, provided that the function of reducing the amount of intracellular cAMP by binding to dopamine D4 receptor protein is not impaired. The modification may be an amino acid sequence in which amino acids are substituted, deleted or inserted at a known functional domain or at a site other than a known functional domain of the dopamine D4 receptor protein, and any number of 1 to 20, preferably 1 to 5, and more preferably 1 to 3 amino acids are deleted, substituted, inserted and/or added. The DNA encoding the dopamine D4 receptor having a modified amino acid sequence is generally synthesized by a conventional site-directed mutagenesis and PCR method, based on a DNA having an inherent base sequence.

In a preferred embodiment, the amino acid sequence of the dopamine D4 receptor protein is shown as SEQ ID NO. 3, and the polynucleotide sequence encoding the dopamine D4 receptor protein in (a) is SEQ ID NO. 1 or has at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 1. For example, when one or more codons are optimized at SEQ ID NO. 1, the optimized polynucleotide sequence will have at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 1.

The CRE transcriptional regulatory element can control the expression of downstream genes, is a convergence point of a plurality of extracellular and intracellular signals, can have known mutation, and does not limit the source of the CRE transcriptional regulatory element in any way.

As a luciferase, the present invention preferably allows for the easy measurement of signals such as transcription, translation, and ultimately activity occurring during the assay by operably linking the CRE transcription regulatory element to the luciferase gene, thereby allowing for rapid indication. The luciferase gene may be included in the construct in the form of a fusion gene, for example, fused to a gene that includes a desired transcription regulatory sequence or exhibits other desired properties.

Taking the dopamine D4 receptor as an example, dopamine signaling is believed to be mediated via cAMP, protein kinase a (pka), and cAMP response element binding protein (CREB) pathways. Dopamine binding to dopamine D4 receptor inhibits intracellular adenylyl cyclase activity, resulting in a decrease in cAMP levels, which decreases PKA phosphorylation, followed by a decrease in phosphorylation of the substrate CREB, a member of the large family of basic leucine zipper (bZIP) domain DNA-binding proteins. Transcription and expression of the luciferase gene, which is eventually operably linked via CRE, is reduced, thereby suppressing the level of fluorescent signal in the final assay system. In this process CREB ultimately makes the cell lines of the invention play a key role by switching dopamine signals to regulate luciferase gene expression.

For the polynucleotide sequence of (b) encoding luciferase whose expression is controlled by CRE transcription regulatory element in the present invention, it can be determined based on known information. The present invention is not limited in any way as to the source, and any commercially available product can be used.

In a preferred embodiment, the amino acid sequence of the luciferase is as shown in SEQ ID NO. 4, and in (b) the polynucleotide sequence encoding the luciferase whose expression is controlled by the CRE transcription regulatory element is SEQ ID NO. 2 or has at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 2. For example, when one or more codons are optimized in SEQ ID NO. 2, the optimized polynucleotide sequence will have at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 2.

For the cell lines of the invention, eukaryotic cells are commonly used as host cells, either from primary cells or from transformed and/or immortalized cell lines, any cell known to those of ordinary skill in the art to alter cAMP levels due to the aforementioned receptors.

The term "host cell" refers to a prokaryotic or eukaryotic cell capable of replicating a vector and/or expressing a heterologous gene encoded by the vector. The term "vector" is used to refer to a vector nucleic acid molecule into which a nucleotide sequence can be inserted for introduction into a cell that can be replicated. The nucleotide sequence may be foreign, meaning that it is foreign to the cell into which the vector is introduced or homologous to sequences in the cell but is not normally found in the host cell nucleic acid at the location of the sequence. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Some of these vectors are commercially available or can be constructed by standard recombinant techniques by those skilled in the art.

Cell lines particularly suitable for the present invention are preferably mammalian cells, more preferably human or rodent cells. Among these cells, CHO cells are more preferably selected from the viewpoint of reducing background signals. Furthermore, CHO-K1 cells are preferred from the viewpoint of functional expression and high expression of the dopamine D4 receptor protein encoded by the polynucleotide (a), i.e., from the viewpoint of achieving a higher signal window.

For the dopamine D4 receptor of the present invention, Forskolin (FSK) is preferably used, an adenylate cyclase agonist, which pre-activates cells to stimulate adenylate cyclase for endogenous cAMP production and thus increase the signal window, however, it was experimentally found that cAMP response after activation depends on the adenylate cyclase isoform present in a specific cell or cell line, and the inventors have verified that HEK-293 cells are not suitable for the present invention, as they may be related to the expression of adenylate cyclase isoform.

In addition to the polynucleotide (a) and polynucleotide (b) sequences described above, other sequences may be included in the polynucleotide (a) and/or polynucleotide (b) of the cell line of the present invention, including, for example, Myc tag, GST tag, His tag, etc., which facilitate selection of purified and cultured cells by introducing the polynucleotide (b) and/or polynucleotide (a) containing such a resistance gene into cells.

In a preferred embodiment, the cell line has the deposit number: CCTCC NO: C2021273.

Construction of cell lines

The method for introducing the polynucleotide (a) and the polynucleotide (b) into the host cell may be any known method, including, for example, transfection, calcium phosphate method, electroporation, particle bead bombardment, method using viruses, etc., and the selection of a specific method generally depends on the type of cell to be transformed, and the specific environment in which the transformation is carried out; the cell line of the present invention is preferably transfected, and more preferably selected from any one of electroporation transfection, lipofection, and calcium ion transfection. After the introduction of the polynucleotide (a) and the polynucleotide (b) into the cell, the cell is usually cultured at a normal culture temperature of 37 ℃ for a period of usually 1 to 24 hours.

The state after introduction is not limited to this, and may be in the form of a plasmid in the cell or may be directly integrated into the genome. The transferred polynucleotide (a) and polynucleotide (b) may be on the same plasmid or on different plasmids, which also contain genes encoding resistance drugs, and the transfected cells are selected by adding the corresponding resistance drugs.

In a second aspect, the invention provides a method for constructing the cell line, preferably comprising the steps of: (1) transfecting a host cell with a polynucleotide sequence encoding dopamine D4 receptor protein to produce a transformed host cell, and (2) overexpressing a CRE-fused luciferase plasmid in the transformed host cell.

The above-mentioned "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.

In step (1) of the method for constructing a cell line of the present invention, the obtained transformed host cell may be prepared by transfection or may be commercially available; for example, commercially available transformed host cells of this type include, but are not limited to: CHO-K1/D4 from Perkinelmer, and the like.

In step (2) of the method for constructing a cell line of the present invention, the CRE-fused luciferase plasmid can be prepared by a conventional method, or can be obtained commercially; for example, commercially available host cells for such transformation include, but are not limited to: CS186804 pNL (NlucP/CRE/Hygro) from Promega, CRE/CREB Reporter Kit from BPS bioscience, etc., preferably CS186804 pNL (NlucP/CRE/Hygro) from Promega.

In a third aspect, the invention provides the use of the cell line described above for detecting a small molecule compound, polypeptide, protein, glycan, nucleic acid, monoclonal antibody or neutralizing antibody, Fc fusion protein or a combination thereof selected against the dopamine D4 target.

The small molecule compound, polypeptide, protein, glycan, nucleic acid, monoclonal antibody or neutralizing antibody thereof and Fc fusion protein aiming at the dopamine D4 target can be naturally occurring or artificially manufactured; may be biologically active or biologically inactive, as long as it is any drug candidate that can be used for testing and screening the dopamine D4 target.

As small molecule compounds, agonists, antagonists, inverse agonists, superagonists, forward allosteric effectors, reverse allosteric effectors, silent allosteric effectors, allosteric ligands or the like are included.

The antibodies include anti-dopamine D4 receptor antibodies PA5-102663, 2B9/DRD4, ab20424 or NLS 3921.

The agonist includes A-412997, ABT-724, or PD-168077.

The antagonist comprises L-745870 or PNU-101387G.

The invention also provides application of the cell line in detecting and preventing and/or treating the neuropsychiatric diseases related to the dopamine D4 receptor of the mammal.

In some embodiments, the neuropsychiatric disease is schizophrenia, schizophrenic disorders, acute schizophrenia, chronic schizophrenia, NOS schizophrenia, schizoid personality disorder, schizotypal personality disorder, delusional disorder, psychosis, psychotic disorder, brief psychotic disorder, shared psychotic disorder, psychotic disorder due to a physical disorder, drug-induced psychosis, psychoaffective disorder, aggression, confusion, parkinson's disease, irritable psychosis, tourette's syndrome, organic or NOS psychosis, epilepsy, agitation, post-traumatic stress disorder, behavioral disturbance, neurodegenerative disorder, alzheimer's disease, parkinson's disease, movement disorder, huntington's disease, dementia, affective disorder, anxiety disorder, affective psychosis, depression, major depressive disorder, depression disorder, anxiety disorder, psychosis, or a combination thereof, Mood disorders, bipolar disorder, mania, seasonal affective disorder, attention deficit hyperactivity disorder, obsessive compulsive disorder, vertigo, epilepsy, pain, neuropathic pain susceptibility states, inflammatory pain, fibromyalgia, migraine, cognitive impairment, movement disorders, restless leg syndrome, multiple sclerosis, sleep disorders, sleep apnea, narcolepsy, excessive daytime sleepiness, jet lag, drowsiness side effects of medications, insomnia, substance abuse dependence, addiction, eating disorders, sexual dysfunction, hypertension, emesis, Lesche-Nyhane disease, Wilson's disease, autism, Huntington's chorea and premenstrual dysphoria.

Detection method

The fourth aspect of the present invention provides a method for detecting biological activity of a dopamine D4 target compound, which comprises the following steps: (1) preparing samples with different concentration gradients, and contacting the samples with the cell lines respectively; (2) determining changes in luciferase levels expressed by each cell line; (3) and calculating the biological activity of the test sample, wherein the test sample is a dopamine D4 target candidate drug solution.

The biological activity of the test sample reflects the function of the test sample on the dopamine D4 target, wherein the test sample can be an agonist or an antagonist or any inactive candidate drug to be detected.

The dopamine D4 receptor is similar to dopamine D3 receptor and belongs to Gi-coupled GPCR (G protein-coupled receptor), and two methods for detecting GPCR signals are mainly used at present, namely detecting the cAMP level in cells and detecting the instant release of calcium flow in cells. For Gi-coupled GPCR receptors, G α 15 protein is often overexpressed in cell lines overexpressing the GPCR, a Gq-like signaling pathway is constructed in the cell, and the transient release of calcium flux is detected to determine whether the GPCR is agonized or antagonized.

The inventor found in the research of constructing a cell line with the biological activity of a dopamine D3 target compound that G alpha 15 protein is over-expressed in a host cell transfected with D3, and the activity of a candidate drug is detected by detecting the intracellular calcium flow, but unfortunately, the inventor has not succeeded; when the inventors over-expressed the CRE-fused luciferase plasmid into a host cell transfected with dopamine D3 receptor, particularly when CHO-K1 cells which can stably express dopamine D3 receptor and luciferase whose CRE transcription regulatory element controls the expression are constructed, by detecting the level of cAMP in cells, the experimental result shows that the cell line has a higher signal window, meanwhile, the method has better stability and repeatability, and can be used for accurately detecting the bioactivity of the dopamine D3 receptor stimulant, in the light of the above experiments, the inventors further tried to construct CHO-K1 cells stably expressing dopamine D4 receptor and luciferase expressed under the control of CRE transcription regulatory element in order to obtain the expected effect, and the results show that the cell line can be successfully constructed, the constructed cell line has good function, and the activity detection result of the dopamine D4 target compound is accurate.

Before starting detection, the constructed cell line can be cultured, so that the cell generation number is between 2 and 4; then, the cells of P2-P4 are planted in a cell culture plate, and the number of the cells is controlled to be 10000-20000 cells/hole, preferably 20000 cells/hole. The material and type of the cell culture plate used in the incubation process can be adjusted according to the needs, and the invention is not limited in any way. Typically, a standard 384-well plate can be used, with 20 μ L of cell suspension per well; the incubation temperature was 37 ℃.

In step (1) of the detection method of the present invention, samples of different concentration gradients are prepared and contacted with the cell line of the present invention; preferably, the test sample is a dopamine D4 target candidate drug solution, the concentration gradient is 2-20 times of dilution gradient, and 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times and the like can be selected.

In a preferred embodiment, in the step (1), after the test article is sufficiently contacted with the cell line, the cell line is further treated by adding forskolin solution. The sufficient contact is to allow the candidate drug to be sufficiently combined with the cell line receptor for a certain time, and the contact time is not too short, and usually the contact time is not less than 10min, preferably 10-30 min, such as 10min, 15min, 20min, 25min, etc.

Forskolin (FSK for short) is an activator of adenylate cyclase, and is added for the purpose of pre-activation to stimulate adenylate cyclase and generate cAMP, so that the increase of endogenous cAMP level is beneficial to increase of a signal window.

In a preferred embodiment, the final concentration of the forskolin solution in step (1) is 0.1 to 0.5 μ M, such as 0.12, 0.15, 0.18, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, etc.;

preferably, the final concentration of the forskolin solution is 0.1-0.3 mu M; more preferably, the final concentration of the forskolin solution is 0.15-0.25 μ M; even more preferably, the final concentration of the forskolin solution is 0.2 μ M.

Through research, the inventor finds that the concentration of forskolin solution directly influences the cAMP level in cells to further cause the change of a signal window, and shows a remarkably enhanced signal window when the final concentration of forskolin solution is 0.1-0.3 μ M, more preferably 0.15-0.25 μ M, and even more preferably 0.2 μ M. The following is speculated through experiments: for example, cells that target the D4 receptor, if forskolin is present in too high a concentration, the activation of adenylate cyclase will exceed the inhibition of adenylate cyclase by agonists, indicating no signal window. Similarly, if forskolin concentration is too low, such that intracellular cAMP levels are too low, the cAMP-lowering effect of the compound will not be apparent. The final concentration refers to the final concentration at the end of the experimental run.

In a preferred embodiment, the step (2) is to determine the change of the luciferase level expressed by each cell line of the test group by using a luciferase assay kit, and comparing the change with a positive control group; wherein the positive control group is different from the test group in that the dopamine D4 target candidate drug solution of the test product is replaced by dopamine solution.

In a preferred embodiment, the detection method further comprises setting a High Control (HC) and a Low Control (LC), the High Control (HC) is set to add forskolin solution, and the Low Control (LC) is set to add forskolin and dopamine mixed solution.

It should be understood that the volumes of the solutions added in the High control and Low control groups should be consistent, and solutions of different concentrations are usually prepared with buffer solutions.

In some embodiments, the final concentration of the forskolin solution in each of the High control and Low control groups may be any concentration, preferably 0.1 to 0.5. mu.M, more preferably 0.1 to 0.3. mu.M, more preferably 0.15 to 0.25. mu.M; still more preferably 0.2. mu.M.

In some embodiments, the final concentration of the dopamine solution in the Low control is any concentration after the agonist effect reaches 100%, preferably ≧ 1 μ M, more preferably 1-10 μ M, such as 1 μ M, 2 μ M, 5 μ M, 10 μ M, and the like.

When determining cAMP levels for cell lines, the following set of four different test conditions can be used for testing:

the first group is a positive control group, which contains dopamine and forskolin solution, and is used as a control product for testing the effectiveness of the experiment and also used as a control for the detection result of the test group. The second group was the test group, containing the drug candidate and forskolin solution. The first group and the second group are correspondingly provided with a plurality of parallel experiments according to the prepared concentration gradient of the dopamine and the candidate drug solution, and dose effect curves of the positive control group and the test group are obtained by fitting according to test results so as to calculate the biological activity of the positive control group and the test group. In the first group and the second group, the concentration gradient of the dopamine and the candidate drug solution is 2-20 times of dilution gradient, and usually 3 times, 5 times, 8 times, 10 times and the like can be selected; the final concentration of the forskolin solution in the first and second groups is 0.1 to 0.5. mu.M, preferably 0.1 to 0.3. mu.M, more preferably 0.15 to 0.25. mu.M, and still more preferably 0.2. mu.M.

The third and fourth groups are conventional controls that are used to reflect the signal window and to calculate the relative chemiluminescent units of the drug candidates. The third group is High control, containing forskolin reagent, which is used to increase endogenous cAMP levels. The fourth group was Low control, containing forskolin and dopamine solution. Wherein in the third and fourth groups, the final concentration of the forskolin solution is 0.1-0.5. mu.M, preferably 0.1-0.3. mu.M, more preferably 0.15-0.25. mu.M, and still more preferably 0.2. mu.M; the final concentration of dopamine in the fourth group is more than or equal to 1 μ M, preferably 1-10 μ M.

In a fifth aspect, the present invention provides a method for detecting biological activity of a dopamine D4 target compound, comprising the steps of:

(1) the CHO-K1 cells which co-express dopamine D4 receptor and CRE transcription regulation element to control expressed luciferase are planted in a cell culture plate, and the number of the cells is controlled to be 10000-20000 per hole;

(2) respectively preparing a positive reference substance and a test solution, and setting a dilution concentration gradient, wherein the positive reference substance is a dopamine solution;

(3) adding the dopamine solution and the test solution prepared in the step (2) into the cells, incubating in a cell incubator at 37 ℃ for 15-30 min, adding forskolin solution with the final concentration of 0.1-0.3 mu M, and placing the cells in the cell incubator at 37 ℃ for further incubation for 3-6 h; in the detection process, High control is set to be added with 0.1-0.3 mu M forskolin solution, and Low control is set to be added with 0.1-0.3 mu M forskolin and dopamine solution more than or equal to 1 mu M;

(4) detecting the change of luciferase level in cells by using a luciferase detection kit, reading corresponding chemiluminescence units by using a microplate reader, obtaining a dose effect curve of candidate drugs by fitting by taking the concentration of the dopamine D4 target candidate drugs as abscissa and taking the relative chemiluminescence units as ordinate, and calculating the bioactivity of the candidate drugs.

Specifically, the method for detecting the biological activity of the dopamine D4 target compound is preferably used, and comprises the following steps:

(1) the method comprises the following steps of (1) planting CHO-K1 cells which co-express dopamine D4 receptor and CRE transcription regulatory element to control expressed luciferase into a cell culture plate, wherein the number of the cells is controlled to be 10000-20000/hole, for example 20000/hole;

(2) respectively preparing a positive reference substance and a test solution, and setting a dilution concentration gradient, wherein the positive reference substance is a dopamine solution;

(3) allowing the cells to grow overnight, adding the dopamine solution and the test solution prepared in the step (2) into the cells, incubating in a 37 ℃ cell incubator for 15min, adding forskolin solution with the final concentration of 0.2 mu M, and placing the cells in the 37 ℃ cell incubator for further incubation for 4 h; in the detection process, High control is set to be added with 0.2 mu M forskolin solution, and Low control is set to be added with 0.2 mu M forskolin and 1-10 mu M dopamine solution;

(4) detecting the change of luciferase level in cells by using a luciferase detection kit, reading corresponding chemiluminescence units by using a microplate reader, obtaining a dose effect curve of candidate drugs by fitting by taking the concentration of the dopamine D4 target candidate drugs as abscissa and taking the relative chemiluminescence units as ordinate, and calculating the bioactivity of the candidate drugs.

The relative chemiluminescence units described above are expressed as% activity, which is calculated by the following formula: activity%_g-R_l)/(R_h-R_l) 100, wherein R_gChemiluminescent units, R, of the test set determined by the microplate reader_lChemiluminescent unit of Low control, R, as determined by microplate reader_hThe chemiluminescence unit of High control determined by a microplate reader. It will be appreciated that in the case of parallel sets of tests, R in the formula_g、R_l、R_hThe average value of the test results of each group is shown.

The data analysis is completed by the software of graphpad prism 8.0, the software is used for carrying out four-parameter fitting on the relative chemiluminescence unit and the concentration of the candidate drug to obtain a dose-effect curve, and the biological activity EC of the candidate drug is obtained by calculation₅₀。

The positive progress effects of the invention are as follows:

the cell line can be used for quickly, accurately and efficiently detecting the biological activity of the dopamine D4 target therapeutic drug. The detection method has the characteristics of good specificity, strong specificity, high accuracy, simple and quick operation, high signal window, good repeatability and the like. In addition, the detection method does not need complex instruments or operation steps, and can completely meet the requirement of detecting the biological activity of the small-molecule compound aiming at the dopamine D4 receptor.

Biological material preservation information

The hamster ovary cells (CHO-K1) of the present invention have been deposited in China Center for Type Culture Collection (CCTCC) at 28/9/2021, and the deposition address is: wuhan university in Wuchang Lojia mountain, Wuhan city, Hubei province, China, a postcode: 430072, with the preservation number: CCTCC NO: c2021273, culture name Chinese hamster ovary cells CHO-K1/D4/Nlucp/CRE.

Drawings

FIG. 1 is a diagram: dose-effect profiles of DA-D4R compounds.

Detailed Description

The present invention is described in detail below with reference to examples, which are provided only for the purpose of further illustrating the present invention and are not to be construed as limiting the scope of the present invention, and the insubstantial modifications and adaptations thereof by those skilled in the art based on the above disclosure are within the scope of the present invention.

The experimental reagents, consumables and instruments in the experimental examples are as follows without further description:

1) experimental reagent

2) Experiment consumable

Name (R)	Batch number	Manufacturer of the product
			ProxiPlate-384 Plus	8310-19411	PerkinELmer

3) Laboratory apparatus

Name (R)	Model number	Manufacturer of the product
			Cell counter	BioTech Countstar	Countstar
Milli-Q	IQ 7000	Millipore
			Centrifugal machine	TDZ5-WS	Cence
Envision	2105	PerkinElmer

4) Experimental cells/plasmids

Cell/plasmid name	Batch number	Manufacturer of the product
			CHO-K1	NA	NA
D4 lentivirus	NA	Heyuanbio
			Nlucp/CRE/Hygro	CS186804	Promega

Example 1

Example 1 was designed for functional validation of HEK293/D4 cells.

1.1 purpose of the experiment

HEK293/D4 cells were constructed and validated for their activated cAMP response.

1.2 Experimental procedures

First, construct HEK293/D4 cell

(1) HEK293 cells were trypsinized and plated into 6-well plates at 1X10 per well⁵Placing the cells at 37 ℃ and 5 ℃％CO₂Growing overnight in a cell culture box;

(2) taking a tube of D4 receptor lentivirus out of liquid nitrogen, placing the tube on ice for thawing, and instantly separating at 1000rpm after thawing to enable the solution adhered to the tube wall to sink to the bottom of the tube; the appropriate infection MOI of HEK293 cells was 5, and the required lentivirus volume was calculated from the number of plated cells of the cells and the lentivirus titer;

(3) removing the HEK293 cells from the cell incubator, replacing fresh growth medium for the HEK293 cells, and then adding a volume of D4 receptor lentivirus to the medium to bring the MOI to 5; gently shaking the 6-well plate to distribute the lentiviruses evenly, and then placing the 6-well plate in a cell culture box to continue to grow overnight; the next day, the cells were replaced with fresh growth medium, and then placed in an incubator for cell growth for 24 h;

(4) taking out HEK293 cells from the incubator, and observing the cell state under a mirror; screening was performed using growth medium containing the screening antibiotic (here the screening antibiotic is puromycin at a concentration of 0.6. mu.g/mL); then, the screening culture containing antibiotics is replaced every day until all cells which are not infected with the D4 receptor lentivirus are killed, and the rest cells are cells infected with the D4 receptor lentivirus (namely HEK293/D4 pool cells); HEK293/D4 pool cells were expanded and then functionally validated.

II, functional effect verification of HEK293/D4 cells

(1) Experiment buffer preparation: by ddH₂O, diluting 5 × stimulation buffer 1(cAMP Gi Dynamic kit) into 1 × stimulation buffer, adding IBMX with the final concentration of 0.5mM, and uniformly mixing for later use;

(2) thawing frozen HEK293/D4 cells in a water bath at 37 ℃, adding the cell suspension into a centrifuge tube containing 10mL of HBSS buffer solution, and centrifuging at 1000rpm for 5 min;

(3) discarding the supernatant, resuspending the precipitate with an appropriate amount of experimental buffer, and counting 20 μ L with a cell counter;

(4) taking a proper amount of HEK293/D4 cell suspension to dilute until the final cell amount is 2000 cells/hole;

(5) 4 experimental groups are arranged in total, the first two groups are respectively added with stimulation buffers, and the second two groups are respectively added with dopamine with the same volume and the final concentration of 10 mu M; centrifuging at 1000rpm for 1min, and incubating for 15min with a sealing plate;

(6) the forskolin sets 2 different concentrations and transfers to the experimental plate, the final concentration is 0.5 and 0.25 MuM respectively; centrifuging at 1000rpm for 1 min; in summary, in the 4 experimental groups, the former two groups were added with forskolin with different concentrations, and the latter two groups were added with forskolin with different concentrations and 10 μ M dopamine;

(7) incubating the sealing plate for 45min at room temperature;

(8) add 5. mu.L of cAMP-d2 solution (stock solution diluted 1:20 in lysis buffer) to the plate and centrifuge at 1000rpm for 1 min;

(9) then 5. mu.L of cAMP Eu-cryptate reagent solution (stock solution is diluted by lysis buffer solution according to the ratio of 1: 20) is added into the experiment plate, and the experiment plate is centrifuged for 1min at 1000 rpm;

(10) placing the experimental plate at room temperature and incubating for 1h in a dark place;

(11) reading a plate by using an Envision microplate reader to obtain an HTRF signal; excitation light 320nm, emission light 620nm and 665 nm.

1.3 results and analysis

This experiment found that HEK293/D4 cells produced higher levels of cAMP under forskolin stimulation, while addition of forskolin and dopamine increased cAMP concentration in contrast to the function of the D4 receptor, suggesting that HEK293 cells did not have a normal cAMP response to the dopamine D4 receptor protein, demonstrating that HEK293/D4 cells are not suitable.

Example 2

Experimental example 2 was designed to verify the functional effects of CHO-K1/D4 cells.

2.1 purpose of the experiment

The cAMP response after its activation was verified using cryopreserved CHO-K1/D4 cells.

2.2 Lentiviral information

Lentiviral names	Name of Gene	GenBank ID	Suppliers of goods
				D4 lentivirus	DRD4	NM_000797.4	Heyuanbio

2.3 Experimental procedures

First, CHO-K1/D4 cell is constructed

(1) Cell plating: taking out the cells from the incubator, wiping the surface of the culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; using a pipette pump to suck out the culture medium, slowly adding 5mL of PBS to the wall of the culture medium, and gently shaking up; absorbing PBS, adding 1mL of 0.05% pancreatin, and incubating at 37 ℃ for 3 min; the morphology of the cells was observed under a microscope and digestion was completed when the cells were all rounded. Adding 5mL of growth medium to terminate digestion, gently blowing off cells by using a pipettor, and transferring the cell suspension into a 15mL centrifuge tube; remove 20. mu.L of cell suspension and count on a cell counter. According to 1 × 10⁵Cell/well Density cells were seeded into 24-well plates with 1mL culture medium per well and shaken gently; standing at room temperature for 30-60 min, and then placing in 5% CO₂Culturing overnight in an incubator at 37 ℃;

(2) lentivirus infection: a tube of D4 receptor lentivirus (100. mu.L/tube) was removed from the-80 ℃ freezer, thawed on ice, and after thawing the lentivirus was placed in a centrifuge for flash separation to allow the lentivirus liquid on the tube wall to sink to the bottom of the tube. The literature reports that CHO-K1 cells are suitably infected at a multiplicity of 20, and the required lentivirus volume is calculated from the multiplicity of infection (MOI), lentivirus titer and cell density. Taking out the cells from the incubator, wiping the surface of the culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; replacement ofFresh growth medium, 1mL per well. The prepared lentivirus was then added to the wells, while a negative control well was set, to which no lentivirus was added. Labeling on the plate lid, and placing the cells in 5% CO₂Culturing overnight in an incubator at 37 ℃;

(3) and (3) resistance screening: screening with medium containing F12+ 10% FBS + 5. mu.g/mL Puromycin; and (3) when all the cells in the negative control wells die, the cells in the infected lentivirus wells are pool cells, the pool cells are digested by pancreatin and then are subcultured to a 10cm culture dish for continuous culture, and subsequent functional verification is carried out.

Second, functional effect verification of CHO-K1/D4 cell

(1) Experiment buffer preparation: by ddH₂O, diluting the 5 × stimulation buffer into 1 × stimulation buffer, adding IBMX with the final concentration of 0.5mM, and uniformly mixing for later use;

(2) thawing frozen CHO-K1/D4 cells in a water bath at 37 ℃, adding the cell suspension into a centrifuge tube containing 10mL of HBSS buffer solution, and centrifuging at 1000rpm for 5 min;

(3) discarding the supernatant, resuspending the precipitate with an appropriate amount of experimental buffer, and counting 20 μ L with a cell counter;

(4) taking a proper amount of CHO-K1/D4 cell suspension to dilute the suspension until the final cell amount is 5000 cells/hole;

(5) setting 8 experimental groups in total, wherein the first four groups are respectively added with stimulation buffers, and the second four groups are respectively added with dopamine with the same volume and the final concentration of 1 mu M; centrifuging at 1000rpm for 1min, incubating for 15min with a sealing plate, and paralleling each group for 3 times;

(6) 4 different concentrations of forskolin are set, and the forskolin is transferred to an experimental plate respectively, and the final concentrations are 2.5, 1.5, 1.0 and 0.5 mu M respectively; centrifuging at 1000rpm for 1 min; in summary, the former four groups of the 8 experimental groups were added with forskolin of different concentrations, and the latter four groups were added with forskolin of different concentrations and 1 μ M dopamine;

(7) incubating the sealing plate for 45min at room temperature;

(8) add 5. mu.L of cAMP-d2 solution (stock solution diluted 1:20 in lysis buffer) to the plate and centrifuge at 1000rpm for 1 min;

(9) then adding 5 mu L of Anti-cAMP-Cryptate solution (stock solution is diluted by lysis buffer solution according to the ratio of 1: 20) into the experimental plate, and centrifuging for 1min at 1000 rpm;

(10) placing the experimental plate at room temperature and incubating for 1h in a dark place;

(11) reading a plate by using an Envision microplate reader to obtain an HTRF signal; excitation light 320nm, emission light 620nm and 665 nm.

2.4 results and analysis

TABLE 2 cAMP response of CHO-K1/D4 cells under dopamine agonism

Note: FSK in the table indicates forskolin, DA indicates dopamine, HTRF signal reflects cAMP concentration, and a larger HTRF signal value indicates a lower cAMP concentration, and vice versa.

From the results, CHO-K1/D4 cells produced high concentrations of cAMP under forskolin stimulation, but the addition of forskolin and dopamine did not significantly inhibit cAMP production, and the signal window was very small, indicating that CHO-K1/D4 cells could only obtain a very weak cAMP response of the D2 receptor protein, which would result in inaccurate detection of the biological activity of the compound, demonstrating that the CHO-K1/D4 cells are not suitable.

Example 3

Test example 3 was designed to validate the functional effect of CHO-K1/D4/Nlucp/CRE cells.

3.1 purpose of the experiment

A CHO-K1/D4 cell line stably expressing Nlucp/CRE was constructed and verified for cAMP response after activation.

3.2 cellular information

Cell name	Growth medium	Algebra
			CHO-K1	F12，10％FBS	P2

3.3 Experimental procedures

Firstly, CHO-K1/D4/Nlucp/CRE cell is constructed

(1) Cell plating: taking out the cells from the incubator, wiping the surface of the culture dish by using 75% alcohol, and placing the culture dish in a biological safety cabinet; using a pipette pump to suck out the culture medium, slowly adding 5mL of PBS to the wall of the culture medium, and gently shaking up; absorbing PBS, adding 1mL of 0.05% pancreatin, and incubating at 37 ℃ for 3 min; the morphology of the cells was observed under a microscope and digestion was completed when the cells were all rounded. Adding 5mL of growth medium to terminate digestion, gently blowing off cells by using a pipettor, and transferring the cell suspension into a 15mL centrifuge tube; remove 20. mu.L of cell suspension and count on a cell counter. According to 1 × 10⁵Cell/well Density cells were seeded into 24-well plates with 1mL culture medium per well and shaken gently; standing at room temperature for 30-60 min, and then placing in 5% CO₂Culturing overnight in an incubator at 37 ℃;

(2) lentivirus infection: a tube of D4 receptor lentivirus (100. mu.L/tube) was removed from the-80 ℃ freezer, thawed on ice, and after thawing the lentivirus was placed in a centrifuge for flash separation to allow the lentivirus liquid on the tube wall to sink to the bottom of the tube. The literature reports that CHO-K1 cells are suitably infected at a multiplicity of 20, and the required lentivirus volume is calculated from the multiplicity of infection (MOI), lentivirus titer and cell density. Taking out the cells from the incubator, wiping the surface of the culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; fresh growth medium was replaced, 1mL per well. The prepared lentivirus is then added to the well while setting a negativeControl wells to which no lentivirus was added. Labeling on the plate lid, and placing the cells in 5% CO₂Culturing overnight in an incubator at 37 ℃;

(3) and (3) resistance screening: screening with medium containing F12+ 10% FBS + 5. mu.g/mL Puromycin; when the cells in the negative control wells all die, the cells in the infected lentivirus wells are pool cells, the pool cells are digested by pancreatin and then are subcultured in a culture dish of 10cm for continuous culture, and luciferase plasmids are transfected on the basis of the cells.

(4) Luciferase plasmid transfection: the cell plating procedure was as described in (1), in which case the CHO-K1 cells were replaced with the selected CHO-K1/D4 pool cells. After the cells grow overnight, taking out the cells from the incubator, wiping the surface of a culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; replace fresh growth medium at 700 μ L per well; transfection systems were formulated using Lipo3000 transfection reagents, as shown in the following table:

after incubation with transfection reagent was completed, it was added to the designated cell wells, while negative control wells were set, to which only growth medium was added and no plasmid transfection was performed. Labeling on the plate lid, and placing the cells in 5% CO₂Culturing overnight in an incubator at 37 ℃;

(5) and (3) resistance screening: screening with a medium containing F12+ 10% FBS + 400. mu.g/mL HygroB + 5. mu.g/mL Puromycin; CHO-K1/D4/Nlucp/CRE pool cells were obtained, which were trypsinized and then passaged to 10cm dishes for further culture for functional validation.

Second, functional effect verification of CHO-K1/D4/Nlucp/CRE cells

(1) Cell plating: taking the CHO-K1/D4/Nlucp/CRE cells out of the incubator, wiping the surface of the culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; the cells were trypsinized and counted, and the cells were individually diluted to 0.75X 10 using growth medium without antibiotics⁶one/mL and 1X10⁶cells/mL, then the cells were plated in a volume of 20. mu.L per wellSeeded to 384 Top-read plates (i.e. 15000 and 20000/well), the cell plates were placed in 5% CO for a 750rpm transient₂Culturing overnight in an incubator at 37 ℃;

(2) adding 1 mu M dopamine solution into a cell plate by using Tecan, centrifuging the cell plate for 1min at 1000rpm, incubating the cell plate with cells at 37 ℃ for 15-30 min, and continuously adding forskolin with preset different concentrations of 0.2, 0.3, 0.4 and 0.5 mu M into the cell plate, wherein 2 multiple holes are respectively arranged. All the treatment groups were Low Control (LC) and High Control (HC) was set. The High control processing step is different from the Low control in that no dopamine solution is added, and the rest is the same as the Low control;

(3) the cell plate was placed continuously in 5% CO after a 1000rpm flash₂And continuously incubating for 4 hours at 37 ℃, then balancing the cell plate for 10-15 min at room temperature, detecting the luciferase level change in the cells by using a luciferase detection kit (Promega, the cargo number is N1110), and reading the corresponding chemiluminescence unit by using an enzyme-labeling instrument.

3.4 results and analysis

Table 3 a: cAMP response in CHO-K1/D4/Nlucp/CRE (20000/well) cells at different concentrations of forskolin

Table 3 b: cAMP response in CHO-K1/D4/Nlucp/CRE (15000/well) cells at different concentrations of forskolin

Note: in the table FSK denotes forskolin and DA denotes dopamine; the signal window is obtained by the following calculation formula: signal window ═ R_h/R_lIn the formula, R_hIs a microplate readerChemiluminescent units, R, of the High control assayed_lChemiluminescent units of Low Control (LC) as determined by microplate reader.

From the results, CHO-K1/D4/Nlucp/CRE cells are successfully constructed in the experiment, and the signal window values of the cells obtained under stimulation of forskolin with different concentrations are about 2 times, which indicates that the CHO-K1/D4/Nlucp/CRE cells have normal cAMP response of dopamine D2 receptor proteins, and the cells can be used for detecting dopamine D4 target compounds. In addition, the optimization test on forskolin concentration and cell density verifies that forskolin is 0.2 mu M, and the optimal signal window can be obtained when the cell density is about 20000.

Example 4

Test example 4 an accuracy test experiment designed for the measurement of the biological activity of dopamine D4 target compounds on CHO-K1/D4/Nlucp/CRE cells was performed.

4.1 purpose of the experiment

The biological activity evaluation of the dopamine D4 target compound is carried out by using cryopreserved CHO-K1/D4/Nlucp/CRE cells, namely, the agonistic activity of DA-D4R on a D4 receptor is detected, and the feasibility and the accuracy of the method are evaluated.

4.2 cellular information

4.3 Experimental procedures

(1) Cell plating: taking the CHO-K1/D4/Nlucp/CRE cells out of the incubator, wiping the surface of the culture dish by 75% alcohol, and placing the culture dish in a biological safety cabinet; the cells were trypsinized and counted, and the cells were diluted to 1X10 cells using growth medium without antibiotics⁶cells/mL, then 20. mu.L per well of the cell plate into 384 top reading plate (i.e. 20000/well), 750rpm of the cell plate after transient exposure to 5% CO₂Culturing overnight in an incubator at 37 ℃;

(2) preparing 7 DA solutions with different concentrations by using the highest point DA concentration of 1 μ M and the dilution multiple of 4 times;

(3) adding pre-set DA solutions of different concentrations to the cell plate using Tecan, starting at 1. mu.M, diluting 4-fold, for 7 concentration points, subsequently centrifuging the cell plate at 1000rpm for 1min, and after incubating the DA compounds with the cells at 37 ℃ for 15min, continuing to add forskolin to the final concentration of 0.2. mu.M in the cell plate; the High Control (HC) and Low Control (LC) groups were set up simultaneously according to the procedure of example 3, wherein the High Control (HC) was added with forskolin solution only to a final concentration of 0.2. mu.M, and the Low Control (LC) was added with forskolin solution 0.2. mu.M and DA 1. mu.M.

(4) The cell plate was placed in 5% CO continuously after 750rpm flash₂Continuously incubating for 4 hours at 37 ℃, then balancing for 10-15 min at room temperature, detecting the luciferase level change in the cells by using a luciferase detection kit (Promega, cat # N1110), reading corresponding chemiluminescence units by using an enzyme-labeling instrument, obtaining a dose-effect curve of the candidate drug by fitting with the concentration of DA as an abscissa and the relative chemiluminescence unit as an ordinate, and calculating the biological activity EC of the candidate drug₅₀。

4.4 results and analysis

The DA-D4R dose-effect curve obtained by fitting the test is shown in figure 1; table 4 shows the accuracy evaluation results of the method for detecting the bioactivity of dopamine D4 target drug, where the activity% represents relative chemiluminescence unit and is calculated by the following formula: activity%_g-R_l)/(R_h-R_l) 100, wherein R_gChemiluminescent units, R, of the test set determined by the microplate reader_lChemiluminescent units of Low Control (LC) as determined by microplate reader, R_hChemiluminescent units of High Control (HC) as determined by the microplate reader. The data analysis is completed by the software of graphpad prism 8.0, the concentration of DA is used as the abscissa, the relative chemiluminescence unit is used as the ordinate, the dose-effect curve (shown in figure 1) is obtained by four-parameter fitting of the relative chemiluminescence unit and the concentration, and the software analyzes and calculates to obtain the biological activity EC of DA₅₀。

Table 4: accuracy evaluation result of dopamine D4 target drug bioactivity detection method

From the results in the table and fig. 1, it can be seen that the agonistic activity EC50 value of DA at the D4 receptor is consistent with the literature report, which indicates that the CHO-K1/D4/Nlucp/CRE cell line has higher accuracy and can meet the detection requirement of the dopamine D4 target compound.

Example 5

Example 5A repeated assay designed to test the biological activity of dopamine D4 target compounds on CHO-K1/D4/Nlucp/CRE cells.

5.1 Experimental purposes frozen CHO-K1/D4/Nlucp/CRE cells were used to assay for dopamine D4 target compound bioactivity and to evaluate the reproducibility of the method.

5.2 cellular information

5.3 Experimental procedures

The same experimental procedure as in example 4 was used for the dopamine agonist and Brilaroxazine (RP5603) to repeat the procedure at different time intervals and to calculate the EC of the dopamine agonist and Brilaroxazine₅₀(ii) a Wherein the Brilaroxazine has the following structural formula:

5.4 results and analysis

Table 5: repeated evaluation result of dopamine D4 target drug bioactivity detection method

Experiment time	DA-D4R EC50Value of	Brilaroxazine EC50Value of
			20210915	158.5nM	NR
20210918	NR	4241nM
			20210923	166.3nM	4168nM

Note: NR in the table indicates that the item was not tested.

From the results, the EC of compound dopamine and RP5603 was determined several times₅₀The value is consistent with the value reported in the literature and the repeatability is good, which shows that the CHO-K1/D4/Nlucp/CRE cell has stable function and good repeatability, and the cell can be used for accurately and rapidly detecting the dopamine D4 target candidate drug.

SEQUENCE LISTING

<110> Shanghai Pivot Biotech Co., Ltd

NHWA PHARMA. Corp.

<120> cell lines and methods for detecting biological activity of dopamine D4 target compound

<130> P21018259C

<160> 4

<170> PatentIn version 3.5

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cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg cagaccgtga 2100

tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg ctgtgggccg 2160

aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac aacgtcctga 2220

ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc ggggacagtc 2280

agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg gagcagcaga 2340

ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg cgagcctaca 2400

tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac ttcgacgatg 2460

ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc accgtcggtc 2520

gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc gaggtgctgg 2580

ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta ggtcgagttt 2640

aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac atctgtgtgt 2700

tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag ccttcaaccc 2760

agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt 2820

ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg 2880

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 2940

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 3000

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 3060

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 3120

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 3180

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 3240

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 3300

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 3360

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 3420

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 3480

gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 3540

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 3600

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 3660

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 3720

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 3780

agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt accagtgctt 3840

gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag tggcctgact 3900

ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca gcgcagcaat 3960

gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc agccagcagg 4020

gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt ctatgagctg 4080

ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag ttgtggccat 4140

tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca actctggttc 4200

ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag tcagctcctt 4260

agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca tggtaatggc 4320

agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg tgaccggcga 4380

gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct cttgcccggc 4440

gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca tcatcgggaa 4500

tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca gttcgatata 4560

gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg tttcggggtg 4620

tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac gaaaatgttg 4680

gatgctcata ctcgtccttt ttcaatatta ttgaagcatt tatcagggtt actagtacgt 4740

ctctcaagga taagtaagta atattaaggt acgggaggta ttggacaggc cgcaataaaa 4800

tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat 4860

acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg 4920

caagtgcagg tgccagaaca tttctct 4947

<210> 3

<211> 419

<212> PRT

<213> Homo sapiens

<400> 3

Met Gly Asn Arg Ser Thr Ala Asp Ala Asp Gly Leu Leu Ala Gly Arg

1 5 10 15

Gly Pro Ala Ala Gly Ala Ser Ala Gly Ala Ser Ala Gly Leu Ala Gly

20 25 30

Gln Gly Ala Ala Ala Leu Val Gly Gly Val Leu Leu Ile Gly Ala Val

35 40 45

Leu Ala Gly Asn Ser Leu Val Cys Val Ser Val Ala Thr Glu Arg Ala

50 55 60

Leu Gln Thr Pro Thr Asn Ser Phe Ile Val Ser Leu Ala Ala Ala Asp

65 70 75 80

Leu Leu Leu Ala Leu Leu Val Leu Pro Leu Phe Val Tyr Ser Glu Val

85 90 95

Gln Gly Gly Ala Trp Leu Leu Ser Pro Arg Leu Cys Asp Ala Leu Met

100 105 110

Ala Met Asp Val Met Leu Cys Thr Ala Ser Ile Phe Asn Leu Cys Ala

115 120 125

Ile Ser Val Asp Arg Phe Val Ala Val Ala Val Pro Leu Arg Tyr Asn

130 135 140

Arg Gln Gly Gly Ser Arg Arg Gln Leu Leu Leu Ile Gly Ala Thr Trp

145 150 155 160

Leu Leu Ser Ala Ala Val Ala Ala Pro Val Leu Cys Gly Leu Asn Asp

165 170 175

Val Arg Gly Arg Asp Pro Ala Val Cys Arg Leu Glu Asp Arg Asp Tyr

180 185 190

Val Val Tyr Ser Ser Val Cys Ser Phe Phe Leu Pro Cys Pro Leu Met

195 200 205

Leu Leu Leu Tyr Trp Ala Thr Phe Arg Gly Leu Gln Arg Trp Glu Val

210 215 220

Ala Arg Arg Ala Lys Leu His Gly Arg Ala Pro Arg Arg Pro Ser Gly

225 230 235 240

Pro Gly Pro Pro Ser Pro Thr Pro Pro Ala Pro Arg Leu Pro Gln Asp

245 250 255

Pro Cys Gly Pro Asp Cys Ala Pro Pro Ala Pro Gly Leu Pro Arg Gly

260 265 270

Pro Cys Gly Pro Asp Cys Ala Pro Ala Ala Pro Ser Leu Pro Gln Asp

275 280 285

Pro Cys Gly Pro Asp Cys Ala Pro Pro Ala Pro Gly Leu Pro Pro Asp

290 295 300

Pro Cys Gly Ser Asn Cys Ala Pro Pro Asp Ala Val Arg Ala Ala Ala

305 310 315 320

Leu Pro Pro Gln Thr Pro Pro Gln Thr Arg Arg Arg Arg Arg Ala Lys

325 330 335

Ile Thr Gly Arg Glu Arg Lys Ala Met Arg Val Leu Pro Val Val Val

340 345 350

Gly Ala Phe Leu Leu Cys Trp Thr Pro Phe Phe Val Val His Ile Thr

355 360 365

Gln Ala Leu Cys Pro Ala Cys Ser Val Pro Pro Arg Leu Val Ser Ala

370 375 380

Val Thr Trp Leu Gly Tyr Val Asn Ser Ala Leu Asn Pro Val Ile Tyr

385 390 395 400

Thr Val Phe Asn Ala Glu Phe Arg Asn Val Phe Arg Lys Ala Leu Arg

405 410 415

Ala Cys Cys

<210> 4

<211> 171

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids of luciferase

<400> 4

Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala

1 5 10 15

Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu

20 25 30

Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu

35 40 45

Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr

50 55 60

Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys

65 70 75 80

Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr

85 90 95

Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe

100 105 110

Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr

115 120 125

Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu

130 135 140

Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val

145 150 155 160

Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala

165 170

Claims (11)

1. A cell line for detecting the biological activity of a dopamine D4 target compound, comprising: (a) a polynucleotide encoding dopamine D4 receptor protein; and (b) a polynucleotide encoding a luciferase whose expression is controlled by a CRE transcription regulatory element.

2. The cell line according to claim 1, wherein the cell line is a mammalian cell, preferably a CHO cell; more preferably CHO-K1 cells.

3. The cell line of claim 2, wherein the amino acid sequence of the dopamine D4 receptor protein is set forth in SEQ ID NO. 3, the amino acid sequence of the luciferase is set forth in SEQ ID NO. 4;

preferably, in (a), the sequence of the polynucleotide encoding dopamine D4 receptor protein is SEQ ID NO 1; and/or (b), the sequence of the polynucleotide for coding the luciferase controlled by the CRE transcription regulatory element to be expressed is SEQ ID NO. 2.

4. The cell line of any one of claims 1 to 3, wherein the cell line has a accession number of CCTCC NO: C2021273.

5. Use of a cell line according to any one of claims 1 to 4 for detecting one or a combination of small molecule compounds, polypeptides, proteins, glycans, nucleic acids, monoclonal antibodies or neutralizing antibodies, Fc fusion proteins selected against the dopamine D4 target.

6. Use of the cell line of any one of claims 1 to 4 for the detection of a medicament for the prevention and/or treatment of a neuropsychiatric disorder associated with the dopamine D4 receptor in a mammal.

7. A method for detecting the biological activity of a dopamine D4 target compound, which is characterized by comprising the following steps: (1) preparing test samples with different concentration gradients, and respectively contacting the test samples with the cell line of any one of claims 1-4; (2) determining changes in luciferase levels expressed by each cell line; (3) and calculating the biological activity of the test sample, wherein the test sample is a dopamine D4 target candidate drug solution.

8. The assay of claim 7, wherein in step (1), the cell line is further treated with forskolin solution after the test sample has been brought into sufficient contact with the cell line.

9. The detection method according to claim 8, wherein the forskolin solution has a final concentration of 0.1 to 0.5 μ M;

preferably, the final concentration of the forskolin solution is 0.1-0.3 mu M; more preferably, the final concentration of the forskolin solution is 0.15-0.25 μ M; even more preferably, the final concentration of the forskolin solution is 0.2 μ M.

10. The assay of claim 7, wherein the step (2) is a step of measuring changes in the level of luciferase expressed by each cell line of the test group using a luciferase assay kit and comparing the measured changes with a positive control group; wherein the positive control group is different from the test group in that the dopamine D4 target candidate drug solution of the test product is replaced by dopamine solution;

preferably, the method further comprises setting a high control and a low control, wherein the high control is set by adding forskolin solution, and the low control is set by adding forskolin and dopamine mixed solution;

more preferably, the number of cells in the cell line is 10000-20000 cells/well, preferably 20000 cells/well.

11. The detection method according to claim 10, wherein in the step (2), after the test sample is contacted with the cell line, the test sample is incubated in an incubator at 37 ℃ for 15-30 min, then forskolin solution is added, and the test sample is placed in the incubator at 37 ℃ for further incubation for 3-6 h; and/or, the high control in the detection process is set to be added with forskolin solution with the final concentration of 0.1-0.5 mu M, preferably 0.1-0.3 mu M, more preferably 0.15-0.25 mu M, and further more preferably 0.2 mu M; and/or, the low control in the detection process is set as adding 0.1-0.5 μ M, preferably 0.1-0.3 μ M, more preferably 0.15-0.25 μ M, and even more preferably 0.2 μ M forskolin solution and dopamine solution with concentration of more than or equal to 1 μ M, preferably 1-10 μ M, and more preferably 1 μ M.

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