CRBN Neo-substrate expression plasmid, cell line and application of CRBN Neo-substrate expression plasmid in PROTAC and / or molecular gel screening

Abstract

The invention discloses a CRBN Neo-substrate expression plasmid, a cell line and an application of the CRBN Neo-substrate expression plasmid and the cell line in PROTAC and / or molecular glue screening. The CRBN Neo-substrate expression plasmid is a plasmid obtained by sequentially inserting a KOZAK nucleic acid sequence behind a CMV (cytomegalovirus) promoter of a pCDNA5-FRT-TO plasmid and fusing a CRBN Neo-substrate nucleic acid sequence with luciferase, and the CRBN Neo-substrate expression plasmid is a plasmid obtained by sequentially inserting a KOZAK nucleic acid sequence behind the CMV promoter of the pCDNA5-FRT-TO plasmid; the CRBN Neo-substate nucleic acid sequence is selected from one or more of the nucleic acid sequences of IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, [delta] NP63a and ZBTB16 (Zinc Boron Boron Nitride 16). The method provided by the invention can be used for high-throughput screening of PROTAC and / or molecular glue with CRBN ligands.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and pharmacy, and particularly relates to a CRBN Neo-substrate expression plasmid, a cell line, and their applications in PROTAC and / or molecular glue screening. Background Art

[0002] Cereblon (CRBN) is part of the E3 ubiquitin ligase system in cells, which can ubiquitinate proteins and then degrade the ubiquitinated proteins through the proteasome. The ubiquitin-proteasome system (UPS) is not only used to destroy abnormal or damaged proteins, but also helps to regulate the levels of some normal proteins. Among them, CRBN is mainly responsible for guiding the ubiquitination of target proteins. The substrate specificity of CRBN is regulated by thalidomide and its derivatives. Currently, the discovered CRBN new substrates (Neo-substrate) include IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1 (CK1α), TAP63a, ΔNP63a, RNF166, RAB28, Aromatase, and ZBTB16 (PLZF) (Anovel cereblon modulatorrecruits GSPT1 to the CRL4(CRBN)ubiquitin ligase.Nature.2016Jul 14;535(7611):252-7;Lenalidomide derivatives and proteolysis-targeting chimeras forcontrolling neosubstrate degradation.Nat Commun.2023Aug18;14(1):4683;Aromatase is a novel neosubstrate of cereblon responsible forimmunomodulatory drug-induced thrombocytopenia.Blood.2020Jun 11;135(24):2146-2158;Thalidomide promotes degradation of SALL4,a transcription factorimplicated in Duane Radial Ray syndrome.Elife.2018Aug 1;7:e38430.doi:10.7554 / eLife.38430).

[0003] In recent years, a new type of protein degradation technology - Proteolysis Targeting Chimeras (PROTAC) has attracted extensive attention. PROTAC is a heterobifunctional molecule with a ligand that binds to the target protein at one end and a ligand that binds to the E3 ubiquitin ligase at the other end, connected by a suitable linker in the middle. The degradation of the target protein by PROTAC is achieved through the ubiquitin-proteasome system: PROTAC binds to the target protein (POI) and the E3 ubiquitin ligase (mainly VHL and CRBN), forming a ternary complex that tags the target protein with ubiquitin. The ubiquitinated protein is recognized and degraded by the 26S proteasome in the cell, thus completely eliminating its function and achieving the purpose of treating diseases. The most commonly used E3 ubiquitin ligases bound by PROTAC are VHL and CRBN. Compared with PROTAC based on VHL, the ligand of the E3 ubiquitin ligase in PROTAC based on CRBN has a smaller molecular weight, is easier to optimize for drugability, and has more advantages in oral absorption. However, CRBN ligands generally have selectivity problems and are prone to off-target effects. Although in some cases, degrading the target protein and Neo-substrate may be beneficial, degrading some Neo-substrates can cause some unwanted toxicities, such as: Ikaros (IKZF1) and Aiolos (IKZF3) are important for lymphocyte development, and the degradation of TAP63a, ΔNP63a, and SALL4 is related to teratogenicity. Therefore, PROTAC designed based on CRBN ligands needs to evaluate its degradation activity against Neo-substrate during the screening stage to avoid unnecessary toxic and side effects to the greatest extent while increasing selectivity.

[0004] Molecular Glue refers to a class of small molecules that can connect more than two proteins in a paste-like form. Different from the ternary bifunctional compound form (bivalent molecule) of PROTAC, Molecular Glue can simultaneously achieve the two functions of recruiting POI and E3 ubiquitin ligase through a short drug fragment (monovalent degrader), and has unparalleled advantages over PROTAC in terms of drugability and other aspects. Currently, most Molecular Glues are discovered accidentally, and most of the existing "rational design" strategies are to optimize the structure and drugability based on CRBN-based Molecular Glue compounds through medicinal chemistry means. However, due to the narrow scope of the structure-activity relationship of CRBN-based Molecular Glue, small changes in key positions will lead to huge changes in activity and targets, so each structure needs to be screened with Neo-substrate for more targeted structure optimization.

[0005] At present, there are mainly two methods for evaluating the selectivity of CRBN ligands, namely Western Blot and proteomics. However, both of these methods have multiple drawbacks. For example, Western Blot has many operation steps, a long cycle, unstable antibodies, poor result repeatability, and high requirements for the operation level of experimental personnel, and it cannot be used for high-throughput screening. Although proteomics can be used for high-throughput screening, the screening cost is high, and it is impossible to ensure consistent labeling efficiency among samples, resulting in large errors in quantitative results, high false positives. At the same time, due to limitations in protein solubility, ionization degree, and poor protein digestion effect, many proteins cannot be accurately quantified. Therefore, establishing a simple, rapid, and high-throughput CRBN ligand selectivity screening scheme is an urgent problem to be solved in current drug development. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a CRBN Neo-substrate expression plasmid, which is a plasmid obtained by sequentially inserting a KOZAK nucleic acid sequence and a luciferase-fused CRBN Neo-substrate nucleic acid sequence after the CMV promoter of the pCDNA5-FRT-TO plasmid; the CRBN Neo-substrate nucleic acid sequence is selected from one or more of the nucleic acid sequences of IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, ΔNP63a, and ZBTB16.

[0007] In some embodiments, the nucleic acid sequence of the CRBN Neo-substrate expression plasmid is selected from one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0008] Another technical problem to be solved by the present invention is to provide a CRBN Neo-substrate expression cell line, which is obtained by integrating the CRBN Neo-substrate nucleic acid sequence in the above-mentioned CRBN Neo-substrate expression plasmid into the cell chromatin by using the FLP-FRT recombination technique.

[0009] In some embodiments, the FLP-FRT recombination technique specifically is: transfecting the CRBN Neo-substrate expression plasmid and the pOG44 plasmid into the 293-FLP-In-T-Rex cell line; the CRBN Neo-substrate expression plasmid has FRT sites.

[0010] In some embodiments, the CRBN Neo-substrate expression plasmid and the pOG44 plasmid are transfected into the 293-FLP-In-T-Rex cell line by liposome transfection.

[0011] In some embodiments, the CRBN Neo-substrate expression plasmid and the pOG44 are transfected into the 293-FLP-In-T-Rex cell line at a ratio of 1:7-10, or 1:8-10, or 1:9.

[0012] In some embodiments, the transfection is co-transfection.

[0013] In some embodiments, the cellular chromatin is the chromatin of the 293-FLP-In-T-Rex cell line.

[0014] In some embodiments, the CRBN Neo-substrate expression cell line is selected from one or more of the cell lines fused with firefly luciferase and the target gene IKZF1, the cell line fused with firefly luciferase and the target gene IKZF2, the cell line fused with firefly luciferase and the target gene IKZF3, the cell line fused with firefly luciferase and the target gene IKZF4, the cell line fused with firefly luciferase and the target gene GSPT1, the cell line fused with firefly luciferase and the target gene SALL4, the cell line fused with firefly luciferase and the target gene CSNK1A1 (CK1α), the cell line fused with firefly luciferase and the target gene TAP63a, the cell line fused with firefly luciferase and the target gene ΔNP63a, and the cell line fused with firefly luciferase and the target gene ZBTB16 (PLZF).

[0015] In some embodiments, the CRBN Neo-substrate expressing cells are selected from one or more of Flp-In T-Rex-293_Luc-IKZF1, Flp-In T-Rex-293_Luc-IKZF2, Flp-In T-Rex-293_Luc-IKZF3, Flp-In T-Rex-293_Luc-IKZF4, Flp-In T-Rex-293_Luc-GSPT1, Flp-In T-Rex-293_Luc-SALL4, Flp-In T-Rex-293_Luc-CSNK1A1, Flp-In T-Rex-293_Luc-TAP63α, Flp-In T-Rex-293_Luc-ΔNP63α, Flp-In T-Rex-293_Luc-ZBTB16;

[0016] Flp-In T-Rex-293_Luc-IKZF1 is a cell line fused with firefly luciferase and the target gene IKZF1, Flp-In T-Rex-293_Luc-IKZF2 is a cell line fused with firefly luciferase and the target gene IKZF2, Flp-In T-Rex-293_Luc-IKZF3 is a cell line fused with firefly luciferase and the target gene IKZF3, Flp-In T-Rex-293_Luc-IKZF4 is a cell line fused with firefly luciferase and the target gene IKZF4, Flp-In T-Rex-293_Luc-GSPT1 is a cell line fused with firefly luciferase and the target gene GSPT1, Flp-In T-Rex-293_Luc-SALL4 is a cell line fused with firefly luciferase and the target gene SALL4, Flp-In T-Rex-293_Luc-CSNK1A1 is a cell line fused with firefly luciferase and the target gene CSNK1A1 (CK1α), Flp-In T-Rex-293_Luc-TAP63α is a cell line fused with firefly luciferase and the target gene TAP63a, Flp-In T-Rex-293_Luc-ΔNP63α is a cell line fused with firefly luciferase and the target gene ΔNP63a, Flp-In T-Rex-293_Luc-ZBTB16 is a cell line fused with firefly luciferase and the target gene ZBTB16 (PLZF).

[0017] The third technical problem to be solved by the present invention is to provide the application of the CRBN Neo-substrate expression plasmid in the screening of PROTAC and / or molecular glue; the CRBN Neo-substrate expression plasmid is a plasmid obtained by sequentially inserting a KOZAK nucleic acid sequence and a luciferase-fused CRBN Neo-substrate nucleic acid sequence after the CMV promoter of the pCDNA5-FRT-TO plasmid; the CRBN Neo-substrate nucleic acid sequence is selected from one or more of the nucleic acid sequences of IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, ΔNP63a and ZBTB16; the E3 ubiquitin ligase ligand connected to one end of the PROTAC is CRBN, and the molecular glue targets CRBN.

[0018] In some embodiments, the nucleic acid sequence of the CRBN Neo-substrate expression plasmid is selected from one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0019] In some embodiments, the application of the CRBN Neo-substrate expression plasmid in the screening of PROTAC and / or molecular glue comprises the following steps: after mixing and culturing a test compound, a Dox solution and a solution containing a CRBN Neo-substrate-expressing cell line, adding a luciferase detection reagent to measure the luminescence intensity, and screening for PROTAC and / or molecular glue according to the luminescence intensity; the CRBN Neo-substrate-expressing cell line contains the CRBN Neo-substrate expression plasmid and is capable of expressing CRBN Neo-substrate. The luciferase detection reagent is luciferin containing a lysis solution, such as Vazyme.

[0020] The fourth technical problem to be solved by the present invention is to provide the application of the CRBN Neo-substrate-expressing cell line in the screening of PROTAC and / or molecular glue, wherein the CRBN Neo-substrate-expressing cell line is the CRBN Neo-substrate-expressing cell line as described in the second technical problem above; the E3 ubiquitin ligase ligand connected to one end of the PROTAC is CRBN, and the molecular glue targets CRBN.

[0021] In some embodiments, the application of the CRBN Neo-substrate expressing cell line in PROTAC and / or molecular glue screening comprises the following steps: after mixing and culturing a compound to be tested, a Dox solution and a solution containing the CRBN Neo-substrate expressing cell line, a luciferase detection reagent is added to measure the luminescence intensity, and PROTAC and / or molecular glue screening is carried out based on the luminescence intensity.

[0022] In some embodiments, the CRBN Neo-substrate expressing cells are selected from one or more of Flp-In T-Rex-293_Luc-IKZF1, Flp-In T-Rex-293_Luc-IKZF2, Flp-In T-Rex-293_Luc-IKZF3, Flp-In T-Rex-293_Luc-IKZF4, Flp-In T-Rex-293_Luc-GSPT1, Flp-In T-Rex-293_Luc-SALL4, Flp-In T-Rex-293_Luc-CSNK1A1, Flp-In T-Rex-293_Luc-TAP63α, Flp-In T-Rex-293_Luc-ΔNP63α, Flp-In T-Rex-293_Luc-ZBTB16; Flp-In T-Rex-293_Luc-IKZF1 is a cell line fused with firefly luciferase and the target gene IKZF1, Flp-In T-Rex-293_Luc-IKZF2 is a cell line fused with firefly luciferase and the target gene IKZF2, Flp-In T-Rex-293_Luc-IKZF3 is a cell line fused with firefly luciferase and the target gene IKZF3, Flp-In T-Rex-293_Luc-IKZF4 is a cell line fused with firefly luciferase and the target gene IKZF4, Flp-In T-Rex-293_Luc-GSPT1 is a cell line fused with firefly luciferase and the target gene GSPT1, Flp-In T-Rex-293_Luc-SALL4 is a cell line fused with firefly luciferase and the target gene SALL4, Flp-In T-Rex-293_Luc-CSNK1A1 is a cell line fused with firefly luciferase and the target gene CSNK1A1 (CK1α), Flp-In T-Rex-293_Luc-TAP63α is a cell line fused with firefly luciferase and the target gene TAP63a, Flp-In T-Rex-293_Luc-ΔNP63α is a cell line fused with firefly luciferase and the target gene ΔNP63a, Flp-In T-Rex-293_Luc-ZBTB16 is a cell line fused with firefly luciferase and the target gene ZBTB16 (PLZF);

[0023] Take 1×10 6Set as the luminescence intensity expression threshold, the final concentrations of Dox for different cell lines are as follows: Flp-In T-Rex-293_Luc-IKZF1 0.5 ng / mL, Flp-In T-Rex-293_Luc-IKZF2 3 ng / mL, Flp-In T-Rex-293_Luc-IKZF3 2 ng / mL, Flp-In T-Rex-293_Luc-IKZF4 0.4 ng / mL, Flp-In T-Rex-293_Luc-GSPT1 0.05 ng / mL, Flp-In T-Rex-293_Luc-SALL4 1 ng / mL, Flp-In T-Rex-293_Luc-CSNK1A1 0.4 ng / mL, Flp-In T-Rex-293_Luc-TAP63α 2 ng / mL, Flp-In T-Rex-293_Luc-ΔNP63α 0.2 ng / mL, Flp-In T-Rex-293_Luc-ZBTB16 1 ng / mL; the degradation rate DR calculation formula is: DR(%) = (luminescence intensity read value of each well of Cpd – Avg_ZPE) * 100 / (Avg_HPE - Avg_ZPE); the luminescence intensity read value of each well of Cpd represents the luminescence intensity measured by adding a luciferase detection reagent after mixing and culturing the test compound, Dox solution, and the solution containing the CRBN Neo-substrate expressing cell line in each well; Avg_HPE represents the average luminescence intensity of the wells replacing the medium containing the CRBN Neo-substrate expressing cell line with the same volume of medium, representing the background signal value when the target completely disappears; Avg_ZPE is the average luminescence intensity of the wells lacking the test compound, representing the maximum signal read value when the target is not degraded at all; variable slope fitting and XL-fit plug-in are used to calculate the DC of the test compound 50 ; According to DC 50 and the maximum degradation efficiency for PROTAC and / or molecular glue screening.

[0024] The beneficial effects of the present invention compared with the prior art are as follows: the CRBN Neo-substrate expression plasmid and CRBN Neo-substrate expression cell line provided by the present invention can perform high-throughput screening on PROTAC and / or molecular glue with CRBN ligands, having the advantages of simplicity, time-saving, real-time monitoring, and high accuracy, and having positive significance for drug research and development and clinical application.

[0025] The following will further illustrate the concept, specific structure, and technical effects of the present invention with reference to the drawings to fully understand the purpose, features, and effects of the present invention. Description of the Drawings

[0026] Figure 1 are the degradation curves of 8 positive molecules against IKZF1;

[0027] Figure 2 are the degradation curves of 8 positive molecules against IKZF2;

[0028] Figure 3 are the degradation curves of 8 positive molecules against IKZF3;

[0029] Figure 4 are the degradation curves of 8 positive molecules against IKZF4;

[0030] Figure 5 are the degradation curves of 8 positive molecules against GSPT1;

[0031] Figure 6 are the degradation curves of 8 positive molecules against SALL4;

[0032] Figure 7 are the degradation curves of 8 positive molecules against CSNK1A1;

[0033] Figure 8 are the degradation curves of 8 positive molecules against TAP63α;

[0034] Figure 9 are the degradation curves of 8 positive molecules against ΔNP63α;

[0035] Figure 10 are the degradation curves of 8 positive molecules against ZBTB16. Detailed implementation manners

[0036] For the convenience of those skilled in the art to understand, some terms appearing in this article are explained and described.

[0037] In this article, the singular forms of "a", "one" and "the" include their plural forms, unless the context otherwise indicates. Therefore, for example, "a reagent" can be understood to include multiple reagent components.

[0038] In this article, unless otherwise specified, the terms "comprising", "including" or "containing" mean containing the listed numerical values, steps or components, but do not exclude the presence of other numerical values, steps or components.

[0039] In this article, A "and / or" B means that it can be any of the three cases: A and B, A alone, and B alone. For example: "Applications in PROTAC and / or molecular glue screening" means applications in PROTAC screening, molecular glue screening, and applications in both PROTAC screening and molecular glue screening.

[0040] In this text, "target point" and "target" have the same meaning.

[0041] In some embodiments of the present invention, the nucleic acid sequence of the CRBN Neo-substrate expression plasmid of the present invention also includes KOZAK nucleic acid sequences and various variants of the luciferase-fused CRBN Neo-substrate nucleic acid sequence. The variants include nucleic acid sequences from the same region that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the genes or regions described herein (i.e., having one or more deletions, insertions, substitutions, reverse sequences, etc.). Therefore, the content of the present invention should be understood to extend to such variants that achieve the same result, even though the actual nucleic acid sequences among individuals have minor genetic variations.

[0042] The inventors used the FLP-FRT recombination technique to insert FRT sites into the genome of HEK-293T cells, enabling them to rapidly integrate the target gene into the cell chromatin through the FRT recombinase (pOG44), achieving rapid insertion and expression of the target gene. Using the FLP-FRT technique, the inventors successfully constructed cell lines of IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1 (CK1α), TAP63a, ΔNP63a, and ZBTB16 (PLZF) fused with firefly luciferase. For the RNF166, RAB28, and Aromatase genes, the inventors also attempted to construct cell lines, but were unable to succeed due to extremely low expression levels.

[0043] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicated by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0044] 1 Construction of Neo-substrate cell lines

[0045] 1.1 Construction of gene expression vector: CRBN Neo-substrate (IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, ΔNP63a and ZBTB16) was cloned into the pCDNA5-FRT-TO plasmid (Invitrogen, V652020, containing the inducible expression regulatory site Tet operate sequence). After the CMV promoter, the KOZAK sequence (enhancing the expression of CRBN Neo-substrate) and the luciferase fusion CRBN Neo-substrate sequence were inserted in turn to obtain the CRBN Neo-substrate plasmid, and their nucleic acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 respectively.

[0046] 1.2 Cell line construction: The CRBN Neo-substrate plasmid and the pOG44 plasmid (carrying the FRT recombinase, Invitrogen, Cat#: V600520) were co-transfected into the 293-FLP-In-T-Rex cell line (Thermo Fisher, Cat#: R78007) at a ratio of 1:9 using the liposome transfection method. Hygromycin was added 48 hours after transfection and continuously cultured to remove negative cells. When the cells could grow stably, it indicated that the stable expression cell line was successfully constructed. The following 10 cell lines were successfully constructed in this invention: Flp-In T-Rex-293_Luc-IKZF1, Flp-In T-Rex-293_Luc-IKZF2, Flp-In T-Rex-293_Luc-IKZF3, Flp-In T-Rex-293_Luc-IKZF4, Flp-In T-Rex-293_Luc-GSPT1, Flp-In T-Rex-293_Luc-SALL4, Flp-In T-Rex-293_Luc-CSNK1A1, Flp-In T-Rex-293_Luc-TAP63α, Flp-In T-Rex-293_Luc-ΔNP63α, Flp-In T-Rex-293_Luc-ZBTB16.Flp-In T-Rex-293_Luc-IKZF1 is a cell line fused with firefly luciferase and the target gene IKZF1, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:1; Flp-In T-Rex-293_Luc-IKZF2 is a cell line fused with firefly luciferase and the target gene IKZF2, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:2; Flp-In T-Rex-293_Luc-IKZF3 is a cell line fused with firefly luciferase and the target gene IKZF3, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:3; Flp-In T-Rex-293_Luc-IKZF4 is a cell line fused with firefly luciferase and the target gene IKZF4, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:4; Flp-In T-Rex-293_Luc-SALL4 is a cell line fused with firefly luciferase and the target gene SALL4, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:5; Flp-In T-Rex-293_Luc-GSPT1 is a cell line fused with firefly luciferase and the target gene GSPT1, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:6; Flp-In T-Rex-293_Luc-CSNK1A1 is a cell line fused with firefly luciferase and the target gene CSNK1A1, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:7; Flp-In T-Rex-293_Luc-TAP63α is a cell line fused with firefly luciferase and the target gene TAP63a, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:8; Flp-In T-Rex-293_Luc-ΔNP63α is a cell line fused with firefly luciferase and the target gene ΔNP63a, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:9; Flp-In T-Rex-293_Luc-ZBTB16 is a cell line fused with firefly luciferase and the target gene ZBTB16, and the nucleic acid sequence of the CRBN Neo-substrate plasmid it uses is as shown in SEQ ID NO:10.Since the FRT sites are fixed, the genomes of the obtained cell lines are completely reproducible. When preparing the cell lines, the inventors set up control cells. So far, no recombination has been found in non-FLP-IN cells, which further proves that the above 10 cell lines can be reproduced without doubt according to the technical solutions disclosed in this embodiment. The control cells are HEK-293 cells without FLP-IN recombination sites. If there is non-specific recombination, the control cells will also survive in the resistance screening stage.

[0047] 1.3 Induced expression of CRBN Neo-substrate: The target cells were induced with gradient-diluted Doxycycline (Dox) for 24 hours, and then luciferin (Vazyme, Cat#: DD1202-01-AA1AB, a luciferase detection reagent) containing lysis buffer was added, and the mixture was incubated with shaking for 15 minutes to fully lyse and mix the cells. The dose-response curve was calculated and fitted. Since the higher the Dox concentration, the more CRBN Neo-substrate is expressed, and the corresponding luminescence intensity is greater. In order to make the CRBN Neo-substrate expression levels of each cell line consistent, 1×10 6 was set as the luminescence intensity expression threshold, and the Dox concentration of each cell line was calculated using Graphpad Prism. The final Dox concentrations of different cell lines are as follows: Flp-In T-Rex-293_Luc-IKZF1 0.5 ng / mL, Flp-In T-Rex-293_Luc-IKZF2 3 ng / mL, Flp-In T-Rex-293_Luc-IKZF3 2 ng / mL, Flp-In T-Rex-293_Luc-IKZF4 0.4 ng / mL, Flp-In T-Rex-293_Luc-GSPT1 0.05 ng / mL, Flp-In T-Rex-293_Luc-SALL4 1 ng / mL, Flp-In T-Rex-293_Luc-CSNK1A1 0.4 ng / mL, Flp-In T-Rex-293_Luc-TAP63α 2 ng / mL, Flp-In T-Rex-293_Luc-ΔNP63α 0.2 ng / mL, Flp-In T-Rex-293_Luc-ZBTB16 1 ng / mL.

[0048] 1.4 Identification of CRBN Neo-substrate expression level by flow cytometry: Cells were collected 24 hours after Dox induction of target cells, fixed with 4% formaldehyde (Themofisher, Cat#: 28908) for 1 hour, incubated with CRBN Neo-substrate antibody at room temperature for 1 hour, resuspended in PBS after centrifugation, incubated with fluorescent secondary antibody for 1 hour, resuspended in PBS after centrifugation, and the fluorescence intensity was measured by flow cytometry. Antibody sources: IKZF1, CST, Cat#: 14859; IKZF2, CST, Cat#: 29360; IKZF3, CST, Cat#: 15103; IKZF4, CST, Cat#: 42427; SALL4, CST, Cat#: 8459; GSPT1, CST, Cat#: 3369; P63, CST, Cat#: 13109; ZBTB16, CST, Cat#: 39784; CSNK1A1, ASSAYPRO, Cat#: 35681-05151; Fluorescent secondary antibody Anti-rabbit IgG(H+L),F(ab')2Fragment(Alexa 488 Conjugate), CST, Cat#: 4412S. The expression levels of 10 cell lines are shown in Table 1:

[0049] Table 1. Expression levels of each target gene after induction

[0050]

[0051] 1.5 Activity assay of positive molecules in degrading CRBN Neo-substrate: The reported molecules were selected as test compounds, and the half-maximal degradation concentration (DC 50 ) and the maximum degradation efficiency (D max ) of positive molecules on CRBN Neo-substrate were measured to determine whether the cell line was successfully constructed.

[0052] The inventors screened for Neo-substrates using 8 CRBN-based PROTACs or molecular glues (abbreviated as positive molecules because they can all degrade the 10 target genes in this example) (CC-885, MCE, Cat#: HY101488; CC-90009, MCE, Cat#: HY-130800; CC-92480, MCE, Cat#: HY-129395; CC-99282, MCE, Cat#: HY-146237; NVP-DKY709, Selleck, Cat#: E1204; TMX-4116, MCE, Cat#: HY145322; Thalidomide, MCE, Cat#: HY-14658; Lenalidomide, Selleck, Cat#: S1029), and used this as an example to demonstrate the advantages of the present invention. The 10 target genes in this example are: IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, ΔNP63a, and ZBTB16.

[0053] 2 Materials

[0054] 2.1 Cells and Culture Media

[0055] The Flp-In T-Rex-293_Luc-IKZF1, Flp-In T-Rex-293_Luc-IKZF2, Flp-In T-Rex-293_Luc-IKZF3, Flp-In T-Rex-293_Luc-IKZF4, Flp-In T-Rex-293_Luc-GSPT1, Flp-In T-Rex-293_Luc-SALL4, Flp-In T-Rex-293_Luc-CSNK1A1, Flp-In T-Rex-293_Luc-TAP63a, Flp-In T-Rex-293_Luc-ΔNP63a, and Flp-In T-Rex-293_Luc-ZBTB16 cell lines constructed by the inventors.

[0056] Culture medium components: Conventional medium (DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 100 μg / mL Hygromycin B), test medium (DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin).

[0057] 2.2 Materials and Reagents (Table 2)

[0058] Table 2, Materials and Reagents

[0059] Name Brand Cat.No. DMEM Life Technologies 10569-010 GlutaMAX Supplement Gibco 35050061 Sodium pyruvate Gibco 11360070 FBS Biosera FB-1058 / 500 Penicillin-Streptomycin HDB HDB prepared HEPES Thermofisher, 15630080 DPBS HDB HDB prepared 0.25% Trypsin-EDTA HDB HDB prepared Hygromycin B Gibco 10687010 Blasticidin S HCl Gibco A1113903 Zeocin Gibco R25005 DMSO Sigma D8418 Doxycycline Sigma 3342-100mg-R Stable-Lite luciferase Assay system Vazyme DD1202-01-AA1AB White, 384well, assay plate Corning 3570 384_LDV plate Labcyte LP-0200

[0060] 1.3 Instruments (Table 3)

[0061] Table 3, Instruments

[0062] Equipment Name Vendor Envision Perkin Elmer Biochemical incubator CIMO <![CDATA[CO2 incubator]]> Thermo Microscope Olymp Cell counter Nexcelom Multidrop Combi Thermo Echo550 LABCYTE dragonfly sptlabtech Bravo Agilent Technologies Plate seal Agilent Technologies Refrigerated Centrifuge Beckman coulter

[0063] 3. Detection Process

[0064] Seed the corresponding cells (4.5×10 6 / dish / 10 mL medium) in a 10 cm petri dish. To make the expression levels of each Neo-substrate consistent, add the corresponding concentration of Dox and incubate in an incubator for 24 hours. The final concentrations of Dox for different cell lines are as follows: Flp-In T-Rex-293_Luc-IKZF1 0.5 ng / mL, Flp-In T-Rex-293_Luc-IKZF2 3 ng / mL, Flp-In T-Rex-293_Luc-IKZF3 2 ng / mL, Flp-In T-Rex-293_Luc-IKZF4 0.4 ng / mL, Flp-In T-Rex-293_Luc-GSPT1 0.05 ng / mL, Flp-In T-Rex-293_Luc-SALL4 1 ng / mL, Flp-In T-Rex-293_Luc-CSNK1A1 0.4 ng / mL, Flp-In T-Rex-293_Luc-TAP63α 2 ng / mL, Flp-In T-Rex-293_Luc-ΔNP63α 0.2 ng / mL, Flp-In T-Rex-293_Luc-ZBTB16 1 ng / mL.

[0065] Pre-dilute the compound to be tested by 3.3 times and serially dilute it 10 times. The maximum final concentration of each compound is shown in Table 4:

[0066] Table 4, Maximum Final Concentrations of Each Compound

[0067]

[0068] Pre-add 25 nL of Dox solution (concentration is 1000 times the Dox final concentration) and 25 nL of the serially diluted compound to be tested (in this example, one of the aforementioned 8 CRBN-based PROTACs or molecular glues, all dissolved in DMSO) in a 384-well plate. The starting concentrations in the cell plate are shown in Table 4. The layout of the 384-well plate is shown in Table 5:

[0069] Table 5, Layout of the 384-Well Plate in This Example

[0070]

[0071]

[0072] Note: HPE represents without cells and without the compound to be tested; ZPE represents with cells but without the compound to be tested; C1, C2, C3, C4, C5, C6, C7, C8 represent the compounds to be tested. -1 to -10 represent different concentration points.

[0073] Add the cell solution with a density of 8000 cells / well / 25 μL of medium. Mix well and centrifuge (1000 rpm, 1 min), and continue to culture and stand in the incubator for 24 hours.

[0074] After 24 hours, take out and add 25 μL of luciferase detection reagent containing lysis buffer (Stable-Lite luciferase Assay system, Vazyme, DD1202-01-AA1AB). After mixing well, measure the luminescence intensity of each well with a microplate reader.

[0075] 4. Data analysis

[0076] The formula for calculating the degradation rate (DR) is: DR(%) = (the reading value of each well of Cpd – Avg_ZPE) * 100 / (Avg_HPE - Avg_ZPE). Avg_HPE is the average luminescence intensity of the wells where 25 μL of medium replaces 8000 cells / well / 25 μL of medium and 25 nL of DMSO replaces the same volume of the compound to be tested dissolved in DMSO (i.e., the average luminescence intensity of the wells without cells and without the compound to be tested), representing the background signal value when the target completely disappears; Avg_ZPE is the average luminescence intensity of the wells where 25 nL of DMSO replaces the same volume of the compound to be tested dissolved in DMSO (i.e., the average luminescence intensity of the wells with cells but without the compound to be tested), representing the maximum signal reading value when the target does not degrade at all. Use variable slope fitting and XL-fit plug-in to calculate the DC of the compound 50 .

[0077] 5. Quality control results

[0078] The quality control results of each cell line are shown in Table 6 below:

[0079] Table 6. Quality control results

[0080] Avg_HPE Avg_ZPE Std_HPE Std_ZPE Z_prime IKZF1 442970 5534080 39200 176707 0.9 IKZF2 251245 1206930 13064 42272 0.8 IKZF3 198160 1915695 13160 43044 0.9 IKZF4 6825 2459365 410 138564 0.8 GSPT1 2490 762495 249 70650 0.7 SALL4 324040 3579935 17884 98391 0.9 CSNK1A1 319415 3797960 17435 99898 0.9 ΔNP63α 533 662125 169 31549 0.9 TAP63α 770 1116893 377 49969 0.9 ZBTB16 773 788108 210 36059 0.9

[0081] The Z values of all test plates are above 0.5, all meeting the requirements.

[0082] 6. Results

[0083] The degradation curves of each compound to be tested are as Figure 1 - Figure 10 shown, and DC 50 and D max are as shown in Table 7 below:

[0084] Table 7, DC 50 and D max

[0085]

[0086]

[0087]

[0088]

[0089] Figure 1 - Figure 10 It shows that each CRBN Neo-substrate cell line has different degrees of response to the positive molecule, indicating that the cell line construction is successful. CC-885 is selected as the positive control molecule for IKZF1, IKZF3, IKZF4, GSPT1, SALL4, CSNK1A1, TAP63α, ΔNP63α and ZBTB16, and NVP-DKY709 is used as the positive control molecule for IKZF2. Eight compounds (CC-885, CC-90009, CC-92480, CC-99282, NVP-DKY709, TMX-4116, Thalidomide, Lenalidomide) are detected by the technology used in the present invention, and the obtained results are consistent with the reports (CC-90009: A Cereblon E3 Ligase Modulating Drug That Promotes Selective Degradation of GSPT1 for the Treatment of Acute Myeloid Leukemia. J Med Chem. 2021 Feb 25;64(4):1835-1843, Table 1. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535,252–257(2016), Figure 1. Model based assessment of food and acid - reducing agent effects on oral absorption of mezigdomide(CC - 92480), a novel cereblon E3 ligase modulator. CPT Pharmacometrics Syst Pharmacol. 2023 Oct;12(10):1473 - 1484, introduction first paragraph. Discovery and characterization of a selective IKZF2 glue degrader for cancer immunotherapy. Cell Chem Biol. 2023 Mar 16;30(3):235 - 247.e12, introduction section. Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT - 2216. J Med Chem. 2022 Jan 13;65(1):747 - 756, Figure 5 )。

[0090] In this article, specific examples are used to elaborate on the principles and implementation methods of the present invention. The description of the above - mentioned embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those of ordinary skill in the art, based on the idea of the present invention, there will be changes in the specific implementation methods and application scopes. It is possible to make changes and improvements to the present invention without exceeding the concept and scope defined by the claims. In summary, the content of the embodiments in this specification should not be construed as a limitation to the present invention.

Claims

1. A CRBN Neo-substrate expression plasmid, characterized in that, The CRBN Neo-substrate expression plasmid is a plasmid obtained by sequentially inserting a KOZAK nucleic acid sequence and a luciferase-fused CRBN Neo-substrate nucleic acid sequence after the CMV promoter of the pCDNA5-FRT-TO plasmid; the CRBN Neo-substrate nucleic acid sequence is selected from one or more of the nucleic acid sequences of IKZF1, IKZF2, IKZF3, IKZF4, SALL4, GSPT1, CSNK1A1, TAP63a, ΔNP63a, and ZBTB16.

2. The CRBN Neo-substrate expression plasmid according to claim 1, wherein The nucleic acid sequence of the CRBN Neo-substrate expression plasmid is selected from one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

3. A CRBN Neo-substrate expressing cell line, characterized in that the CRBN Neo-substrate expressing cell line is obtained by integrating the CRBN Neo-substrate nucleic acid sequence in the CRBN Neo-substrate expression plasmid as described in claim 1 or 2 into the cell chromatin by using the FLP-FRT recombination technique.

4. The CRBN Neo-substrate expressing cell line according to claim 3, characterized in that, The FLP-FRT recombination technique is specifically as follows: transfecting the CRBN Neo-substrate expression plasmid and the pOG44 plasmid into the 293-FLP-In-T-Rex cell line; the CRBN Neo-substrate expression plasmid has an FRT site.

5. The CRBN Neo-substrate expressing cell line according to claim 3, characterized in that, The CRBN Neo-substrate expressing cell line is selected from one or more of a cell line fused with firefly luciferase and the target gene IKZF1, a cell line fused with firefly luciferase and the target gene IKZF2, a cell line fused with firefly luciferase and the target gene IKZF3, a cell line fused with firefly luciferase and the target gene IKZF4, a cell line fused with firefly luciferase and the target gene GSPT1, a cell line fused with firefly luciferase and the target gene SALL4, a cell line fused with firefly luciferase and the target gene CSNK1A1, a cell line fused with firefly luciferase and the target gene TAP63a, a cell line fused with firefly luciferase and the target gene ΔNP63a, and a cell line fused with firefly luciferase and the target gene ZBTB16.

6. Use of the CRBN Neo-substrate expression plasmid in the screening of PROTACs and / or molecular glues, wherein, The CRBN Neo-substrate expression plasmid is the CRBN Neo-substrate expression plasmid as described in claim 1 or 2; the E3 ubiquitin ligase ligand connected to one end of the PROTAC is CRBN, and the molecular glue targets CRBN.

7. Use of the CRBN Neo-substrate expression plasmid according to claim 6 in PROTAC and / or molecular glue screening, characterized in that, Comprising the following steps: After mixing and culturing the compound to be tested, the Dox solution and the solution containing the CRBN Neo-substrate expressing cell line, a luciferase detection reagent is added to measure the luminescence intensity, and PROTAC and / or molecular glue screening is carried out according to the luminescence intensity; the CRBN Neo-substrate expressing cell line contains the CRBN Neo-substrate expression plasmid and is capable of expressing CRBN Neo-substrate.

8. Use of CRBN Neo-substrate expressing cell lines in PROTAC and / or molecular glue screening, wherein, The CRBN Neo-substrate expressing cell line is the CRBN Neo-substrate expressing cell line according to any one of claims 3-5; the E3 ubiquitin ligase ligand connected to one end of the PROTAC is CRBN, and the molecular glue targets CRBN.

9. Use of the CRBN Neo-substrate expressing cell line according to claim 8 in PROTAC and / or molecular glue screening, characterized in that, Comprising the following steps: After mixing and culturing the compound to be tested, the Dox solution and the solution containing the CRBN Neo-substrate expressing cell line, a luciferase detection reagent is added to measure the luminescence intensity, and PROTAC and / or molecular glue screening is carried out according to the luminescence intensity.

10. Use of the CRBN Neo-substrate expressing cell line according to claim 9 in PROTAC and / or molecular glue screening, characterized in that, The CRBN Neo-substrate expressing cells are selected from one or more of Flp-In T-Rex-293_Luc-IKZF1, Flp-In T-Rex-293_Luc-IKZF2, Flp-In T-Rex-293_Luc-IKZF3, Flp-In T-Rex-293_Luc-IKZF4, Flp-In T-Rex-293_Luc-GSPT1, Flp-In T-Rex-293_Luc-SALL4, Flp-In T-Rex-293_Luc-CSNK1A1, Flp-In T-Rex-293_Luc-TAP63α, Flp-In T-Rex-293_Luc-ΔNP63α, Flp-In T-Rex-293_Luc-ZBTB16; Flp-In T-Rex-293_Luc-IKZF1 is a cell line fused with firefly luciferase and the target gene IKZF1, Flp-In T-Rex-293_Luc-IKZF2 is a cell line fused with firefly luciferase and the target gene IKZF2, Flp-In T-Rex-293_Luc-IKZF3 is a cell line fused with firefly luciferase and the target gene IKZF3, Flp-In T-Rex-293_Luc-IKZF4 is a cell line fused with firefly luciferase and the target gene IKZF4, Flp-In T-Rex-293_Luc-GSPT1 is a cell line fused with firefly luciferase and the target gene GSPT1, Flp-In T-Rex-293_Luc-SALL4 is a cell line fused with firefly luciferase and the target gene SALL4, Flp-In T-Rex-293_Luc-CSNK1A1 is a cell line fused with firefly luciferase and the target gene CSNK1A1, Flp-In T-Rex-293_Luc-TAP63α is a cell line fused with firefly luciferase and the target gene TAP63a, Flp-In T-Rex-293_Luc-ΔNP63α is a cell line fused with firefly luciferase and the target gene ΔNP63a, Flp-In T-Rex-293_Luc-ZBTB16 is a cell line fused with firefly luciferase and the target gene ZBTB16; Set 1×10 6 as the luminescence intensity expression threshold. The final concentrations of Dox for different cell lines are as follows: Flp-In T-Rex-293_Luc-IKZF1 0.5 ng / mL, Flp-In T-Rex-293_Luc-IKZF2 3 ng / mL, Flp-In T-Rex-293_Luc-IKZF3 2 ng / mL, Flp-In T-Rex-293_Luc-IKZF4 0.4 ng / mL, Flp-In T-Rex-293_Luc-GSPT1 0.05 ng / mL, Flp-In T-Rex-293_Luc-SALL4 1 ng / mL, Flp-In T-Rex-293_Luc-CSNK1A1 0.4 ng / mL, Flp-In T-Rex-293_Luc-TAP63α 2 ng / mL, Flp-In T-Rex-293_Luc-ΔNP63α 0.2 ng / mL, Flp-In T-Rex-293_Luc-ZBTB16 1 ng / mL; The degradation rate DR calculation formula is: DR(%) = (Cpd reading per well – Avg_ZPE) * 100 / (Avg_HPE - Avg_ZPE); Cpd reading per well represents the luminescence intensity measured by adding a luciferase detection reagent after mixing and culturing the test compound, Dox solution, and the solution containing the CRBN Neo-substrate expressing cell line in each well; Avg_HPE represents the average luminescence intensity of the wells replacing the medium containing the CRBN Neo-substrate expressing cell line with the same volume of medium, representing the background signal value when the target completely disappears; Avg_ZPE is the average luminescence intensity of the wells lacking the test compound, representing the maximum signal reading when the target is not degraded at all; Use variable slope fitting and XL-fit plug-in to calculate the DC 50 ; According to DC 50 and the maximum degradation efficiency for PROTAC and / or molecular glue screening.

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