IL12 / IL23-NanoBiT luciferase report dual system and drug screening method

Abstract

The invention provides an IL12 / IL23-NanoBiT luciferase report dual system and a drug screening method, and belongs to the technical field of drug screening. According to the invention, an IL12 / IL23-NanoBiT luciferase report dual system is constructed, the report system can be applied to detection of generation of heterodimer cell factors IL12 and IL23, and a tool system for simple quantification of generation of IL12 / IL23 is provided. On the basis of verifying that the IL12 / IL23-NanoBiT system can specifically and highly sensitively monitor the assembling formation of IL12 alpha / IL12 beta and IL23 alpha / IL12 beta subunits of cell factors IL12 and IL23, the report system can also screen compounds with influence effects on the assembling and post-translational modification of the IL12 and IL23 subunits, and can be effectively applied to high-throughput screening of the IL12 / IL23 small-molecule inhibitor with high activity and low toxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and specifically relates to an IL12 / IL23-NanoBiT luciferase reporter dual system and a drug screening method. Background Art

[0002] IL12 (subunits consisting of p35 and p40) and IL23 (subunits p19 and p40) are the first cytokines discovered in the IL12 family and have been studied most extensively and deeply. They are clearly shown to play an important role in the pathogenesis of various autoimmune diseases (ADs). Targeting IL12 and IL23 has become an important approach for the treatment of immune-mediated inflammatory diseases. In the clinic, antibody inhibitors that dual-target IL12+IL23 and single-target IL23 have also achieved significant therapeutic effects in AD. The development of IL12 / IL23-related drugs has great value in the alleviation and cure of the disease.

[0003] It is necessary to develop new small molecule inhibitors of IL12 / IL23. Currently, the IL12 / IL23 inhibitors on the market or in preclinical stage are mainly antibody drugs, and the development of related small molecule inhibitors is still blank. Small molecule drugs have a stable chemical structure, and the production conditions are not harsh. In addition, small molecule drugs are suitable for making various dosage forms and can be administered by multiple routes. In general, they are easy to penetrate the cell membrane. Therefore, if small molecule drugs targeting IL12 / IL23 are developed, it will be more conducive to expanding production and benefit more patients. The development of novel and potential IL12 / IL23 small molecule inhibitors can fill the gap in the research field of IL12 / IL23 small molecule inhibitors and will provide a more economical, safe and effective option for the treatment of ADs.

[0004] NanoBiT (NanoLuc Binary Technology) is a highly sensitive luciferase fragment complementation technology that can accurately detect intracellular protein interactions. It has high sensitivity, signal-to-noise ratio and dynamic monitoring range. This technology has been used to monitor membrane protein translocation and secretory protein formation, which shows that NanoBiT technology can be well applied to the study of signal peptide proteins. Therefore, it is feasible to use it to monitor the secretion formation of IL12 / IL23. However, how to achieve this specifically will be a subject that people in this field need to study. Summary of the invention

[0005] The present invention provides an IL12 / IL23-NanoBiT luciferase reporter dual system and a drug screening method. The system can specifically monitor and indicate the generation of IL12 and IL23, and screen out highly effective and low-toxic IL12 / IL23 small molecule inhibitors.

[0006] To achieve the above objectives, the present invention provides an IL12 / IL23-NanoBiT expression vector, characterized in that it includes an IL23-NanoBiT expression vector and an IL12-NanoBiT expression vector containing IL12 / IL23 expression genes and firefly reporter fragments LgBiT and SmBiT, which can specifically monitor and indicate the production of IL12 and IL23.

[0007] Preferably, the IL23 expression genes include IL23α and IL23β genes, and the IL12 expression genes include IL12α and IL12β genes.

[0008] Preferably, the C-terminus of the IL23α gene is connected to the reporter fragment LgBiT, and the C-terminus of the IL12β gene is connected to the reporter fragment SmBiT, and the two pairs are expressed to obtain the IL23-NanoBiT expression vector;

[0009] The C-terminus of the IL12α gene was connected to the reporter fragment LgBiT, and the C-terminus of the IL12β gene was connected to the reporter fragment SmBiT. The two pairs were expressed in pairs to obtain the IL12-NanoBiT expression vector.

[0010] Preferably, the amino acid sequence of IL12α-LgBiT is shown in SEQ ID NO: 1, the amino acid sequence of IL12β-SmBiT is shown in SEQ ID NO: 2; and the amino acid sequence of IL23α-LgBiT is shown in SEQ ID NO: 3.

[0011] Preferably, the backbone vector used in constructing the expression vector is pcDNA3.1(+), which contains a strong promoter CMV to ensure high expression of secretory proteins.

[0012] The present invention also provides an IL12 / IL23-NanoBiT luciferase reporter dual system, comprising an IL23-NanoBiT luciferase reporter system and an IL12-NanoBiT luciferase reporter system, wherein the IL23-NanoBiT luciferase reporter system comprises a cell line for drug screening, an IL23-NanoBiT expression vector according to any of the above technical solutions, a negative control expression vector for verifying the specificity of the system, and an optional positive control drug endoplasmic reticulum stress inducer Tm; the IL12-NanoBiT luciferase reporter system comprises a cell line for drug screening, an IL12-NanoBiT expression vector according to any of the above technical solutions, a negative control expression vector for verifying the specificity of the system, and an optional positive control drug endoplasmic reticulum stress inducer Tm, for high-throughput drug screening of IL12 / IL23 small molecule inhibitors.

[0013] Preferably, the negative control expression vector in the IL23-NanoBiT luciferase reporter system is based on the IL12-NanoBiT expression vector, and a point mutation of IL12β Y246A and IL12α R189A The negative control expression vector in the IL23-NanoBiT luciferase reporter system was obtained by point-mutating IL12β on the basis of the IL23-NanoBiT expression vector. C177R and IL23α C54R get.

[0014] As a preference, only 5,000 live cells are needed for the IL23-NanoBiT luciferase reporter system, and only 10,000 cells are needed for the IL12-NanoBiT luciferase reporter system to achieve 10 6 The luminescence level is high, meeting the requirements of high-throughput screening.

[0015] The present invention also provides a method for high-throughput drug screening, using the IL12 / IL23-NanoBiT luciferase reporter dual system described in any of the above technical solutions, and the screening method is as follows:

[0016] The IL23-NanoBiT luciferase reporter system and the IL12-NanoBiT luciferase reporter system were respectively used for primary screening at a working concentration of 20 μmol / L, and compounds with an inhibition rate greater than 50% were selected for secondary screening;

[0017] The IL23-NanoBiT luciferase reporter system and the IL12-NanoBiT luciferase reporter system were used to rescreen the compounds obtained from the initial screening at a working concentration of 5 μmol / L, respectively, and compounds with cell viability greater than 90% and luciferase inhibition rate greater than 45% were selected to obtain highly active and low-toxic IL12 / IL23 small molecule inhibitors.

[0018] Preferably, the IL12-NanoBiT luciferase reporter system and the IL23-NanoBiT luciferase reporter system only detect the effects of the compounds on the assembly and post-translational modification of IL12 / IL23 heterodimers, but do not affect the assembled mature IL12 and IL23 proteins.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are:

[0020] The present invention constructs an IL12 / IL23-NanoBiT luciferase reporter dual system, and the reporter system can be used to detect the generation of heterodimeric cytokines IL12 and IL23, providing a simple tool system for quantifying IL12 / IL23 generation. On the basis of verifying that the IL12 / IL23-NanoBiT system can specifically and highly sensitively monitor the assembly and formation of IL12α / IL12β and IL 23α / IL12β subunits of cytokines IL12 and IL23, the reporter system can also screen compounds that have an effect on the assembly and post-translational modification of IL12 and IL23 subunits, and can be effectively used in high-throughput screening of IL12 / IL23 small molecule inhibitors with strong activity and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of constructing the IL23-NanoBiT and IL12-NanoBiT reporting systems in Example 1 of the present invention;

[0022] Figure 2 This is a schematic diagram of verifying the specificity of the biological activity signal of the IL23 and IL12 NanoBiT systems in Example 2 of the present invention;

[0023] Figure 3 This is a schematic diagram of verifying the response of the IL12 and IL23 NanoBiT systems to different drugs in Example 3 of the present invention;

[0024] Figure 4 This is a schematic diagram of verifying the high sensitivity of the IL23 and IL12 NanoBiT system responses in Example 4 of the present invention;

[0025] Figure 5 This is a schematic diagram of compounds obtained by multiple rounds of high-throughput screening of the IL23 and IL12 NanoBiT systems in Example 5 of the present invention;

[0026] Figure 6-Figure 15 IL23α-LgBiT, IL23α C54R -LgBiT, IL23α-SmBiT, IL12α-LgBiT, IL12α R189A -LgBiT, IL12α-SmBiT, IL12β-SmBiT, IL12β Y246A -SmBiT, IL12β C177R -Partial sequencing results of SmBiT and IL12β-LgBiT. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Reagents and consumables used in the following examples:

[0029] The solid small molecule compound library was provided by Taoshu Biotechnology Co., Ltd.; the living cell luciferase detection reagent Live Cell Assay System was purchased from Promega; DMEM (high glucose) and fetal bovine serum were purchased from Hyclone Thermo; RNA reverse transcription kit was purchased from Takara; 96-well plate was purchased from Corning; recombinant Anti-IL12B antibody was purchased from Abcam, HA antibody and Tunicamycin reagent were purchased from Cell Signaling Technology; secondary antibodies (including HRP goat anti-mouse and HRP goat anti-rabbit) were purchased from Abcam; NC membrane and ECL developer were purchased from Millipore; BCA protein quantification kit and ampicillin storage solution (100 mg / mL) were purchased from Solarbio; phosphatase inhibitor and protease inhibitor cocktail were purchased from Roche; 5× protein loading buffer, DMSO and other conventional chemical reagents were provided by Shanghai Bioengineering or MacLean; Lipo3000 reagent, eBioscience TM Protein transport inhibitor mixture (500×) was purchased from Invitrogen; Ren Tianqing dry powder was purchased from Sigma-Aldrich, USA. Cell culture dishes and conventional experimental consumables such as centrifuge tubes were provided by Shanghai Bioengineering. Escherichia coli DH5α chemically competent cells were purchased from Nanjing Novozyme. All primers used in the experiment were synthesized by Qingdao Weilai Biotechnology Co., Ltd.

[0030] Example 1 Construction method of IL12 / IL23-NanoBiT drug high-throughput screening system

[0031] S1: Cell culture: Human embryonic kidney cells HEK293T were cultured at 37°C and 5% CO2. DMEM high-glucose medium contained double antibiotics penicillin-streptomycin and 10% fetal bovine serum (FBS).

[0032] S2: Molecular construction: pBiT1.1-C[TK / LgBiT] vector and pBiT2.1-C[TK / SmBiT] vector were purchased from Promega (Madison, WI, USA). PPI MCS Starter System; In order to obtain fusion protein expression vectors with different assembly pairs, the construction is divided into two parts.

[0033] The first step is to design primers to amplify the genes IL12β, IL12α and IL23α. Because the genes in the amplification template do not contain signal peptides, the N-terminal signal peptide sequence corresponding to the gene is introduced through the forward primer, so that the protein subunits assembled to form IL12 and IL23 can be pulled into the endoplasmic reticulum for assembly, folding, subsequent modification and secretion. The tag sequence is introduced at the C-terminus of the gene through the reverse primer to facilitate the subsequent detection of fusion protein expression. The amplified sequence is recombinantly connected between the NheI and XhoI sites of pcDNA3.1(+).

[0034] The second step is to obtain LgBiT and SmBiT gene fragments by digesting the vector pBiT1.1-C[TK / LgBiT] and pBiT2.1-C[TK / SmBiT] with XhoI and XbaI, and then connect and clone them between XhoI and XbaI of the vector constructed in the first step. Since the IL12α gene sequence contains an XbaI site, the construction of IL12α-LgBiT and IL12α-SmBiT is achieved by replacing IL23α in the vector successfully constructed in the second step with IL12α.

[0035] The primer sequences designed to amplify genes IL12β, IL12α and IL23α are shown in Table 1. The underlined sequences are restriction sites, and the lowercase letters are the introduced sequences.

[0036] Table 1 Primer sequences

[0037]

[0038]

[0039] S3: Site-directed mutagenesis: Using the reporter vector of the IL12 and IL23 NanoBiT system as a template, mutate the IL12β, IL12α, and IL23α genes in the vector. Use a point mutagenesis kit to construct the mutation vector, and the specific method is as follows.

[0040] Design of IL12β with mutation sites Y246A and IL12α R189A , IL12β C177R and IL23α C54R The primer sequences are shown in Table 2. The lowercase letters are the mutation site sequences.

[0041] Table 2 Primer sequences

[0042]

[0043]

[0044] Use primers and Phanta Max Super-Fidelity DNA Polymerase in the kit to amplify 1ng of the target plasmid (reaction system 50μL). Add 1μL of DpnI enzyme to the amplified product and digest it at 37℃ for 2h to remove the methylated template plasmid. Take 0.03pmol of the digested product and react at 37℃ for 2h under the catalysis of Exnase II enzyme for vector recombination. The recombinant product is cooled on ice and used to transform competent cells. Pick a single clone on the transformation plate, expand the culture and send the sample culture liquid for sequencing. The engineered strain that has successfully mutated is used to extract the plasmid and freeze it at -80℃ with glycerol.

[0045] S4: Cell transfection: 96-well plate transfection: HEK293T cells at 1.5×10 4 100μL culture medium was plated in a 96-well white plate and cultured for 24h. The culture medium was replaced with fresh serum-free DMEM high-glucose medium 30min before transfection. 2.5ng of paired vectors and 0.01μL of P3000 were added to 5μL serum-free medium, and 0.1μL of Lipo3000 was added to another 5μL serum-free medium. The two dilutions were gently mixed, incubated for 10-15min, and then added to the 96-well white plate and gently mixed. After the cells were cultured for 24h, the live cell luciferase reaction substrate was added to react for 5min, and the luminescence response was detected on the machine. After the detection, the culture medium was aspirated and transferred to a new 96-well white plate, 100μL of sterilized PBS was added to the white plate of the original cultured cells, and the luminescence values ​​of the two white plates were detected again.

[0046] S5: 6-well plate transfection: HEK293T cells were transfected at 5×10 5 2mL culture medium was plated in 6-well plates for 24h. The culture medium was replaced with fresh serum-free DMEM medium 30min before transfection. 1μg of each paired vector and 4μL of P3000 were added to 125μL serum-free medium; 3μL of Lipo3000 was added to another 125μL serum-free medium. The two dilutions were gently mixed, incubated for 10-15min, and then added to the 6-well plate and the culture medium was gently mixed. After the cells were cultured for 6h, the culture medium was replaced with fresh complete medium preheated at 37℃. The culture was continued for 18h, the cells were lysed to collect proteins, and part of the culture supernatant was taken for Western blot detection.

[0047] S6: 15 cm dish transfection: HEK293T cells were cultured for 2 to 3 generations after recovery. When the cells were in good condition, 1×10 7Cells were plated into 15cm dishes, 20mL of culture medium was added to each dish, and cultured for 24h. 30min in advance, the culture medium in the dish was replaced with 20mL of fresh serum-free DMEM high-glucose medium. 2μg of each paired vector and 8μL of P3000 were added to 500μL of serum-free medium, and 5μL of Lipo3000 was added to another 500μL of serum-free medium. The two dilutions were gently mixed, incubated for 10-15min, and then added to the 15cm dish and gently mixed. After 6h, the culture medium was replaced with fresh complete medium preheated at 37℃ and cultured for another 18h. Take a small amount of culture supernatant, add luciferase substrate to detect the luminescence to verify the successful transfection. The cells were then digested and plated into 96-well plates according to the required number.

[0048] S7: Protein extraction and Western blot: Prepare 10% SDS-PAGE gel, 80V, 30min, 120V 1h electrophoresis, after completion, soak the polyacrylamide gel in pre-cooled transfer buffer, and perform sandwich transfer with NC membrane, set constant voltage 100V 1.5h. Use TBST to prepare 5% skim milk powder, carefully take out the transferred NC membrane, protein side up, block at room temperature for 1h; prepare primary antibody according to the instructions, incubate at 4℃ overnight, wash 5 times with TBST, 5min each time; incubate the secondary antibody at room temperature for 1h, wash the PVDF membrane 4 times with TBST, 10min each time. Add ECL development and exposure.

[0049] S8: Compound screening and rescreening: Single well screening: cells were counted at 1.5×10 4 Cells and 100 μL culture medium were plated into 96-well white plates. After 24 hours of culture, each compound was added to the designated wells at 20 mM solution / well. An equal amount of solvent DMSO was used as a blank control well, and 1 μg / mL Tm was used as a positive control. Each well plate was set up with 6 blank wells and 6 positive controls. After 24 hours of incubation, Nano-Glo Live Cell Reagent was added and incubated for 5 minutes before detecting luciferase activity.

[0050] S9: Three-well screening: cells were screened at 1.5×10 4 Each well was plated with 50 μL of culture medium in a 96-well white plate and cultured for 24 hours. Each compound was diluted with fresh complete medium and added to each well at the specified concentration. An equal amount of solvent DMSO was used as a blank, and 1 μg / mL Tm was used as a positive control. The final total volume of culture medium in each well was 100 μL. Each well plate had 6 blank and positive control wells, and 3 wells for each compound. After incubation for 24 hours, the luminescence was detected in the same way as the initial screening.

[0051] S10: Cell activity analysis (Resazurin method): The transfected cells were plated into a 96-well transparent plate in the same way as the rescreening, and the cells were treated with the same dosing method and drug incubation time. 10 μL of 1 mg / mL Resazurin was added to each well and incubated in the dark for 4 hours. The absorbance value of each well was detected using a full-wavelength microplate reader under the conditions of excitation light 544 nm and emission light 595 nm.

[0052] S11: Data calculation and processing: Snapgene software was used to align sequencing data and analyze sequencing results. GraphPad Prism 8 software was used to analyze the detected luciferase chemical response data and plot the graph. The data results are expressed as mean ± standard error (mean ± SEM). The experiments were repeated 3 or more times, and each test was set up with 3 or more replicates. The statistical differences between the two groups were compared using Student's t-test, and the differences between three or more groups of data were compared using One-way ANOVA.

[0053] Example 1 Establishment of efficient IL23-NanoBiT and IL12-NanoBiT reporting systems

[0054] The way LgBiT and SmBiT are assembled with the research protein is one of the key factors affecting the sensitivity of the constructed NanoBiT system. The assembly pairing determines the conformation of the final fusion protein, so it is necessary to detect the response of different assembly expression vectors to determine the best pairing. Since IL12p40, IL12p35 and IL23p19 carry a hydrophobic signal peptide at the N-terminus during translation to guide the newly synthesized protein to transfer to the secretory pathway and be cleaved by signal peptidases in the subsequent process, this study mainly constructs expression vectors in which LgBiT and SmBiT are connected to the C-terminus of each subunit to prevent the reporter protein fragment from being cut and destroyed. The vectors expressing the two subunits of IL12 or IL23 connected with the reporter fragment are paired and transfected into cells respectively. The assembly and combination of IL12p40 and IL12p35 drive the connected LgBiT and SmBiT to combine and emit light. Similarly, the combination of IL12p40 and IL23p19 will cause luminescence. By detecting the chemiluminescent response in the total culture, cells and culture supernatant in the living cell state, the amount of IL12 or IL23 formed can be truly reflected, so that the paired expression vector with the best luminescent response can be selected for subsequent research. The backbone vector pcDNA3.1(+) selected when the system was established contains a strong promoter CMV, which ensures the efficient expression of secretory proteins. It has been proven that when the total amount of vector transfection in a 96-well plate is 5ng / well, nearly 10 5 Order of magnitude response (see methods S2, S4, Figure 1BC, EF). The reporter fragment and the research protein were connected by a 15-amino acid flexible chain GS Linker (GSSGGGGSGGGGSSG) to reduce the steric hindrance generated when the fusion protein was expressed. The ratio of paired expression vectors was 1:1 ( Figure 1 A, D) (see Method S2).

[0055] The chemiluminescent response of HEK293T cells transfected with two IL23α / IL12β pairs for 24 h was detected (see Method S4). The results showed that when IL23α was connected to LgBiT and IL12β was connected to SmBiT, the response effect was much higher than that when IL23α was connected to SmBiT and IL12β was connected to LgBiT. In addition, the response of IL23α-LgBiT / IL12β-SmBiT was mainly caused by IL23 assembled and secreted into the culture supernatant, while only a very low level of luminescence was detected in the cells ( Figure 1 AC). Similar experimental results were observed in the two IL12α / IL12β pairings (see Method S4). The IL12α-LgBiT / IL12β-SmBiT response was better, and the response was mainly caused by IL12 secreted in the culture supernatant ( Figure 1 DF). Therefore, IL23α-LgBiT / IL12β-SmBiT was selected as the IL23-NanoBiT reporter system, and IL12α-LgBiT / IL12β-SmBiT was selected as the IL12-NanoBiT reporter system.

[0056] Example 2 Verification of the specificity of the biological activity signals of the IL23 and IL12 NanoBiT systems

[0057] This project constructs a point mutation IL12β based on IL23 and IL12-NanoBiT expression vectors. Y246A and IL12α R189A , blocking the amino acids necessary for the formation of IL12 heterodimers IL12β Y246 and IL12α R189 The interaction was used as a negative control of the IL12-NanoBiT system. C177R and IL23α C54R , blocking the IL12β necessary for the formation of IL23 heterodimers C177 and IL23α C54The disulfide bond formed by amino acid binding was used as a negative control for the IL23-NanoBiT system. The construction method is shown in S3. In both systems, the response effect and fusion protein expression were detected after equal amounts of vectors were paired or transfected into HEK293T cells alone (see methods S4, S5, and S7). The amino acid sequence and partial sequencing results of the constructed vectors (showing the sequencing peaks of the partial sequence after the start codon and the partial sequence before the stop codon) are shown in Tables 3 and Figure 6-15 .

[0058] The experimental results showed that only when the IL12β and IL12α subunits, and the IL12β and IL23α subunits were expressed in a normal pair, the system could detect a high level of response, and the response was mainly caused by IL12 and IL23 secreted into the supernatant ( Figure 2 A, B). Western blot results showed that the fusion protein expression could be detected in cells and culture supernatant regardless of whether the above vectors were transfected in pairs or alone ( Figure 2 C). Therefore, the experimental results ensure that the luminescent response generated by the IL12-NanoBiT and IL23-NanoBiT systems is specifically caused by the secretion of IL12 and IL23 after formation, respectively, rather than false positive signals generated by the spontaneous association of LgBiT and SmBiT.

[0059] Table 3

[0060]

[0061]

[0062]

[0063] Example 3 Verification of the response of IL12 and IL23-NanoBiT systems to different drugs

[0064] Endoplasmic reticulum stress inducer (tunicamycin, Tm) can inhibit protein N-glycosylation modification and cause the accumulation of unfolded proteins in the endoplasmic reticulum of cells. Protein transport inhibitor (a mixture of Brefeldin A and monensin) can restrict mature secreted proteins to the cell. The two were used to test the changes in the response level of the NanoBiT system when the IL12 / IL23 heterodimer assembly, modification or secretion process was blocked. When 1μg / mL Tm was used for 6h, the inhibition rate of IL23-NanoBiT response was more than 40%, and the inhibition rate of IL12-NanoBiT total response was 50%. The above shows that Tm blocking the assembly and modification of cytokines in the endoplasmic reticulum will inhibit the production of cytokines (see Method S4) ( Figure 3A). When the protein transport inhibitor acted on the system, it was found that the total response level of the IL12-NanoBiT system did not decrease, and the IL23 system was only inhibited at a low level. At this time, the response was mainly caused by cytokines accumulated in the cells. The intracellular response of the IL23 system increased by more than 9 times, and the IL12 system increased by more than 16 times. In contrast, the culture supernatant responses of the IL23 system and the IL12 system were reduced to about 20% of the untreated group (see Method S4) ( Figure 3 B).

[0065] Therefore, these results show that inhibiting the transport of cytokines without blocking the assembly and binding of protein subunits will not have a significant impact on the overall response of the system. That is, the mechanism of the system for subsequent drug screening is mainly to detect the effect of compounds on the assembly and modification of cytokine subunits. At the same time, Tm can serve as an effective positive control drug for the system.

[0066] Example 4 Verification of the High Sensitivity of IL23 and IL12 NanoBiT System Responses

[0067] In high-throughput screening, the sensitivity of the cell model is crucial, so we tested the response effects of different cell numbers in the two systems. The results showed that only 1,000 living cells were needed for each system to produce 10 5 The luminescence level is 10000, and the luminescence level increases steadily with the increase of cell number. The IL23-NanoBiT system only needs 5,000 living cells, while the IL12-NanoBiT system can reach 10 6 Therefore, the dual system has a high sensitivity and only requires a small number of cells to achieve high-throughput screening requirements (see Method S6) ( Figure 4 ).

[0068] Example 5 Verification of the inhibitory effects of compounds obtained by high-throughput screening of IL12 and IL23-NanoBiT dual systems

[0069] The type and quantity of compounds used for high-throughput screening directly determine the probability of obtaining potential pharmacological compounds. Therefore, we selected the Novel Bioactive Compound Library for the physical compound library. The small molecule compounds in it have been preliminarily tested to have biological activity at the cellular level, and the structure is more novel than that of the listed drug library, and it is easier to find active compounds than the drug-like compound library. At the same time, considering that many natural products have been reported to have anti-inflammatory factor effects, some small molecule compounds in the natural product compound library were supplemented and screened. The compound library information is shown in Table 4.

[0070] Table 4

[0071]

[0072] 4119 compounds were screened at a concentration of 20 μmol / L, and compounds with an inhibition rate greater than 50% were selected according to the signal inhibition effect, that is, compounds with Cut off: Log2 (RLU) < -1. 80 compounds with an inhibition rate greater than 50% were screened by the IL23-NanoBiT system, and 18 compounds with an inhibition rate greater than 50% were screened by the IL12-NanoBiT system. These compounds were used for further screening (see Methods S6, S8) ( Figure 5 AB).

[0073] The two systems tested the compounds obtained in the initial screening at a concentration of 5 μmol / L. To improve the accuracy, three replicate wells were set for each compound to be tested. Compounds with cell viability greater than 90% and luciferase inhibition rate greater than 45% were selected (see Method S6, S9) ( Figure 5 CD).

[0074] The inhibitory effect of the compounds obtained by repeated screening on the system response after concentration gradient administration was detected, and the compound information is shown in Table 5. During the detection, the luciferase response was tested in three replicate wells at each concentration, and the cell viability was measured with resazurin at the same time (see Method S6, S10). The average detection value of three wells of each compound at the highest administration concentration of 10 μmol / L is shown in Table 5.

[0075] Table 5

[0076]

[0077]

[0078] In order to deduct the effect of the compounds in Table 5 on cell viability, the (Luc / cellviability)% values ​​of the concentration gradient compounds were used for curve fitting. The results showed that the 8 compounds of the IL23-NanoBiT system (numbered #13, #38, #49, #68, #69, #73, #92, #100) and the 3 compounds of the IL12-NanoBiT system (numbered #20, #24, #73) all had good inhibitory effects, and the luciferase activity (cell viability deducted) inhibition IC50 was below 10 μM ( Figure 5 EF).

Claims

1. An IL12 / IL23-NanoBiT expression vector, characterized in that: The invention comprises an IL23-NanoBiT expression vector and an IL12-NanoBiT expression vector containing IL12 / IL23 expression genes and firefly reporter fragments LgBiT and SmBiT, which can specifically monitor and indicate the production of IL12 and IL23.

2. The expression vector according to claim 1, characterized in that IL23 expression genes include IL23α and IL23β genes, and IL12 expression genes include IL12α and IL12β genes.

3. The expression vector according to claim 2, characterized in that The C-terminus of the IL23α gene was connected to the reporter fragment LgBiT, and the C-terminus of the IL12β gene was connected to the reporter fragment SmBiT, and the two pairs were expressed to obtain the IL23-NanoBiT expression vector; The C-terminus of the IL12α gene was connected to the reporter fragment LgBiT, and the C-terminus of the IL12β gene was connected to the reporter fragment SmBiT. The two pairs were expressed in pairs to obtain the IL12-NanoBiT expression vector.

4. The expression vector according to claim 3, characterized in that The amino acid sequence of IL12α-LgBiT is shown in SEQ ID NO: 1, the amino acid sequence of IL12β-SmBiT is shown in SEQ ID NO: 2; and the amino acid sequence of IL23α-LgBiT is shown in SEQ ID NO:

3.

5. The expression vector according to claim 3, characterized in that The backbone vector used in constructing the expression vector is pcDNA3.1(+), which contains a strong promoter CMV to ensure high expression of secretory proteins.

6. An IL12 / IL23-NanoBiT luciferase reporter dual system, characterized in that: It comprises an IL23-NanoBiT luciferase reporter system and an IL12-NanoBiT luciferase reporter system, wherein the IL23-NanoBiT luciferase reporter system comprises a cell line for drug screening, an IL23-NanoBiT expression vector according to any one of claims 3 to 5, a negative control expression vector for verifying the specificity of the system, and an optional positive control drug endoplasmic reticulum stress inducer Tm; the IL12-NanoBiT luciferase reporter system comprises a cell line for drug screening, an IL12-NanoBiT expression vector according to any one of claims 3 to 5, a negative control expression vector for verifying the specificity of the system, and an optional positive control drug endoplasmic reticulum stress inducer Tm, so as to be used for high-throughput drug screening of IL12 / IL23 small molecule inhibitors.

7. The IL12 / IL23-NanoBiT luciferase reporter dual system according to claim 6, characterized in that: The negative control expression vector in the IL12-NanoBiT luciferase reporter system is based on the IL12-NanoBiT expression vector, and the IL12β Y246A and IL12α R189A The negative control expression vector in the IL23-NanoBiT luciferase reporter system was obtained by point-mutating IL12β on the basis of the IL23-NanoBiT expression vector. C177R and IL23α C54R get.

8. The IL12 / IL23-NanoBiT luciferase reporter dual system according to claim 6, characterized in that: For the IL23-NanoBiT luciferase reporter system, only 5,000 live cells are needed, and for the IL12-NanoBiT luciferase reporter system, only 10,000 cells are needed to achieve 10 6 The luminescence level is high, meeting the requirements of high-throughput screening.

9. A method for high-throughput drug screening, characterized in that: The IL12 / IL23-NanoBiT luciferase reporter dual system according to any one of claims 6 to 8 is used, and the screening method is as follows: The IL23-NanoBiT luciferase reporter system and the IL12-NanoBiT luciferase reporter system were respectively used for primary screening at a working concentration of 20 μmol / L, and compounds with an inhibition rate greater than 50% were selected for secondary screening; The IL23-NanoBiT luciferase reporter system and the IL12-NanoBiT luciferase reporter system were used to rescreen the compounds obtained from the initial screening at a working concentration of 5 μmol / L, respectively, and compounds with cell viability greater than 90% and luciferase inhibition rate greater than 45% were selected to obtain highly active and low-toxic IL12 / IL23 small molecule inhibitors.

10. The method according to claim 9, characterized in that The IL12-NanoBiT luciferase reporter system and the IL23-NanoBiT luciferase reporter system only detect the effects of compounds on the assembly and post-translational modification of IL12 / IL23 heterodimers, but will not affect the assembled mature IL12 and IL23 proteins.

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