CN118603967A - Drug screening method, system and screened drug - Google Patents

Abstract

The present application discloses a drug screening method, system and screened drugs. In the present application, the method includes the steps of S1 respectively constructing a first expression vector containing a first coding sequence and a second expression vector containing a second coding sequence; S2 co-transfecting the first expression vector and the second expression vector into a host cell and culturing the host cell; S3 treating the host cell or its lysate with a candidate drug; S4 adding a luminescent substrate to the host cell or its lysate and then detecting the intensity of the bioluminescent signal, and screening the drug according to the change in the intensity value of the luminescent signal. The high-throughput drug screening system and drug screening method for agonists targeting ARIH1 developed in the present application can directly obtain positive drugs that act on ARIH1 itself, which is of great significance for the development of tumor treatment methods with resistance to drug resistance.

Description

Drug screening method, system and screened drug

Technical Field

The present invention relates to the field of biomedicine, and in particular to a drug screening method, system and screened drugs.

Background Art

Programmed death ligand 1 (PD-L1) is a type I transmembrane protein of 40 kDa that plays an important role in the immune escape of cancer cells. Under normal circumstances, the body's immune system will respond to foreign antigens that accumulate in the lymph nodes or spleen, inducing a large number of antigen-specific killing T cells (CD8+T cells) to proliferate. The combination of PD-L1 and programmed death receptor (PD-1) can transmit inhibitory signals and reduce the proliferation of CD8+T cells in lymph nodes. Therefore, PD-1 and PD-L1 can be used as immunotherapy targets for different types of cancer.

At present, there is drug resistance in the combined treatment of PD1 and PD-L1, so it is urgent to find a treatment plan to overcome drug resistance. In previous studies, the inventors found that ARIH1 is a key regulatory factor in overcoming immune checkpoint blockade (ICB) resistance. See the paper ARIH1 activates STING-mediated T-cell activation and senses tumors to immune checkpoint blockade. Therefore, the construction of a high-throughput drug screening system based on ARIH1 as a target is of great significance for the development of tumor treatment methods with anti-drug resistance. The inventors have previously developed a series of drug screening methods for the ARIH1 target to screen drugs that can affect the expression of ARIH1. However, the drugs screened by this method do not directly act on the protein structure itself, resulting in low screening efficiency. Therefore, there is an urgent need in this field to develop new drug screening methods for the ARIH1 target.

Summary of the invention

The object of the present invention is to provide a drug screening method.

Another object of the present invention is to provide a drug obtained by screening the above method.

In order to solve the above technical problems, the first aspect of the present invention provides a method for screening a drug for tumor immunotherapy, the method comprising the steps of:

S1, constructing a first expression vector containing a first coding sequence and a second expression vector containing a second coding sequence, respectively, wherein the first coding sequence is as shown in SEQ ID NO: 1; and the second coding sequence is as shown in SEQ ID NO: 2;

S2, co-transfecting the first expression vector and the second expression vector into a host cell and culturing the host cell;

S3, treating the host cell or its lysate with a candidate drug; and

S4, adding a luminescent substrate, detecting the luminescent signal intensity of the host cells or their lysate, and screening drugs according to changes in the luminescent signal intensity value.

In some preferred embodiments, the drug is used to reduce resistance to tumor immunotherapy.

In some preferred embodiments, the method comprises The system screens candidate drugs.

In some preferred embodiments, the screening of drugs according to the change in the luminescent signal intensity value comprises the step of: screening drugs that reduce the luminescent signal intensity value of the host cells or their lysates. In some preferred embodiments, the screening of drugs according to the change in the luminescent signal intensity value comprises the step of: if the luminescent signal intensity value of the host cells or their lysates treated with the candidate drug is reduced compared to that without the candidate drug treatment, then the drug is a positive drug.

In some preferred embodiments, the positive drug can reduce the luminescence intensity value of the host cells or their lysates by at least 30%, and there is a significant difference (P<0.05), more preferably by at least 40%, more preferably by at least 50%, for example, 50%, 60%, 70%, 80% or 90%.

In some preferred embodiments, the first expression vector and the second expression vector are co-transfected into the host cell at a ratio of 1:1.

In some preferred embodiments, the first expression vector and the second expression vector are co-transfected into host cells using liposome transfection.

In some preferred embodiments, the first coding sequence encodes LgBit-Ar fusion protein.

In some preferred embodiments, the second coding sequence encodes Af-SmBit fusion protein.

In some preferred embodiments, the LgBit-Ar fusion protein is shown in SEQ ID NO: 3.

In some preferred embodiments, the Af-SmBit fusion protein is as shown in SEQ ID NO:4.

In some preferred embodiments, the first expression vector is a pLgBit-Ar plasmid, and the pLgBit-Ar plasmid is connected to the C-terminus of the LgBit sequence to a polynucleotide sequence encoding the ARIH1 protein subunit Ar.

In some preferred embodiments, the second expression vector is a pAf-SmBit plasmid, and a polynucleotide sequence encoding the ARIH1 protein subunit Af is connected to the N-terminus of the SmBit sequence in the pLgBit-Ar plasmid.

In some preferred embodiments, the luminescent substrate is a luciferase substrate.

In some preferred embodiments, the host cell is a 293T cell.

A second aspect of the present invention provides a drug screening system, the drug screening system comprising:

Reaction module; the reaction module comprises a plurality of reaction chambers, the reaction chambers being used to react host cells or their lysates with candidate drug reaction liquid;

A drug delivery module; the drug delivery module is used to deliver the candidate drug into the reaction chamber of the reaction module;

A signal recognition module; the signal recognition module is used to identify the luminescent signal intensity value of the reaction chamber; and

Signal processing module: The signal processing module is used to receive the luminescent signal intensity value from the signal recognition module and screen positive drugs according to the change of the luminescent signal intensity value.

In some preferred embodiments, the drug screening system is used for high-throughput screening of anti-tumor immune resistance drugs.

In some preferred embodiments, the host cell can co-express LgBit-Ar fusion protein and Af-SmBit fusion protein. More preferably, the host cell contains the first expression vector and the second expression vector.

The third aspect of the present invention provides a method for evaluating the anti-tumor immune resistance of a candidate drug, the method comprising the steps of:

Sa, constructing a host cell, wherein the host cell is capable of expressing LgBit-Ar fusion protein and Af-SmBit fusion protein;

Sb, culturing the host cells, treating the host cells or their lysates with a candidate drug, adding a luminescent substrate, and detecting and recording changes in the luminescence intensity values of the host cells or their lysates;

Sc, when the luminescence intensity value of the host cells or their lysates treated with the candidate drug is reduced (preferably reduced by at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%) compared with the control group not treated with the candidate drug, it is determined that the candidate drug has anti-tumor immune resistance.

In a preferred embodiment, if the decrease in the luminescence intensity value is significantly different, it is determined that the candidate drug has anti-tumor immune resistance.

The fourth aspect of the present invention provides a drug obtained by screening using the method described in the first aspect of the present invention or the drug screening system described in the second aspect of the present invention.

Compared with the prior art, the present invention has at least the following advantages:

(1) The present invention utilizes the characteristics that the regulatory domain Ariadne of ARIH1 binds to and shields the catalytic active domain RING2 of ARIH1 in the autoinhibitory state, while the Ariadne domain dissociates from the RING2 domain and releases the catalytic active site in the activated state to develop a high-throughput drug screening system and a drug screening method for agonists targeting ARIH1. This method can directly obtain positive drugs that act on ARIH1 itself, which is of great significance for the development of tumor treatment methods with resistance to drug resistance.

(2) The drug screening method provided by the present invention is optimized through experiments to obtain a drug that can match the stereochemical structure. The protein structure fragments of the system were identified, and a plasmid combination with high luminescence signal intensity was further screened, which improved the sensitivity and efficiency of the screening method.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

FIG1 is a schematic diagram of the screening principle of ARIH1 allosteric activation according to an embodiment of the present invention;

FIG2 is a diagram showing eight plasmid combinations for ARIH1 allosteric activation screening according to an embodiment of the present invention;

FIG3 is a graph showing the luminescence detection results of eight plasmid combinations according to an embodiment of the present invention;

4 is a schematic diagram of verifying the combination of pLgBit-Ar and pAf-SmBit plasmids by point mutation of the Ar domain according to an embodiment of the present invention;

FIG5 is a result of detecting intracellular ARIH1 allosteric activation of positive drugs according to an embodiment of the present invention;

FIG6 is a result of in vitro ARIH1 allosteric activation detection of positive drugs according to an embodiment of the present invention;

FIG. 7 is a diagram showing the interaction between a positive drug detected by Thermal Shift and ARIH1 protein according to an embodiment of the present invention;

FIG. 8 is a diagram showing the effect of positive drugs on the ubiquitination activity of ARIH1 protein according to an embodiment of the present invention.

DETAILED DESCRIPTION

ARIH1 activates innate immunity through the ARIH1-DNA-PKcs-STING pathway and induces T cell activation, which is an effective mechanism to alleviate ICB resistance. However, in the prior art, the drugs screened by the drug screening method for the ARIH1 target do not directly act on the protein structure itself, and the screening efficiency is low. After extensive and in-depth research, the inventors have developed a method based on The drug screening method based on the technology utilizes the characteristics that in the autoinhibitory state, the regulatory domain Ariadne of ARIH1 protein binds to and shields the catalytic active domain RING2 of ARIH1; in the activated state, the Ariadne domain dissociates from the RING2 domain and releases the catalytic active site. The method can screen drugs that directly act on the ARIH1 protein. The screened drugs promote the allosteric activation of the ARIH1 protein and have the potential to be used as tumor immunity-related drugs.

Drug Screening Methods

The embodiment of the present invention relates to a drug screening method for screening tumor immunotherapy drugs, comprising steps S1-S4,

S1, constructing a first expression vector containing a first coding sequence and a second expression vector containing a second coding sequence, respectively, wherein the first coding sequence is as shown in SEQ ID NO:1; and the second coding sequence is as shown in SEQ ID NO:2;

S2, co-transfecting the first expression vector and the second expression vector into a host cell and culturing the host cell;

S3, treating the host cell or its lysate with a candidate drug; and

S4, adding a luminescent substrate, detecting the luminescent signal intensity of the host cells or their lysate, and screening drugs according to changes in the luminescent signal intensity value.

SEQ ID NO: 1:

atggtcttcacactcgaagatttcgttggggactgggaacagacagccgcctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgctgcagaatctcgccgtgtccgtaactccgatccaaaggattgtccggagcggtgaaaatgccctgaagatcgacatccatgtcatcatcccgtatgaaggtctga gcgccgaccaaatggcccagatcgaagaggtgtttaaggtggtgt accctgtggatgatcatcactttaaggtgatcctgccctatggcacactggtaatcgacggggttacgccgaacatgctgaactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaccccgacggctccat gctgttccgagtaaccatcaacagtgggagttccggtggtggc gggagcggaggtggaggctcgagcagagatgcacaggagcgatctagggcagccctgcagaggtacctgttctactgtaatcgctatatgaaccacatgcagagcctgcgctttgagcacaaactatatgctcaggtgaaacagaaaatggaggagatgcagcagcacaacatgtcctggattgaggtgcagttcctga agaaggcagttgatgtcctctgccagtgtcgtgccacactcatgtaca cttatgtcttcgctttctacctcaaaaagaataaccagtccattatctttgagaataaccaagcagatctagagaatgccacagaggtgctctcgggctaccttgaacgagatatttcccaagattctctgcaggatataaagcagaaagtacaagacaagtacagatactgtgagagtcgacgaagggttttgttacagcatgtgcatgaa ggctatgaaaaagatctgtggggagtacattgaggac

SEQ ID NO:2:

Atggactcggacgagggctacaactacgagttcgacgaggacgaggagtgcagtgaggaggacagcggcgccgaggaggaggaggacgaagacgacgacgagccggacgatgataccctggatctgggcgaggtggagctggtggagcccgggctgggcgtcggcggggagcgggacggactgctgtgcggggagacgggcggtgg cggcggcagcgctctggggcccggcggtggcggcggcggcggcggcggcggtggtggtggcgggccggggcatgagcaggaggaggattaccgctacgaggtgctcacggccg agcagattctacaacacatggtggaatgtatccgggaggtcaacgaggtcatccagaatccagcaactatcacaagaatactccttagccacttcaattggggataaagagaagctaatggaaaggtactttgatggaaacctggagaagctctttgctgagtgtcatgtaattaatccaagtaaaaaagtctcgaacacgccagatgaatacaaggtcatcag cacaggatatgccttgtcagatctgctacttgaactaccctaactcgtatttcactggccttgaatgtggacataagttttgtatgcagtgctggagt gaatatttaactaccaaaataatggaagaaggcatgggtcagactatttcgtgtcctgctcatggttgtgatatcttagtggatgacaacacagttatgcgcctgatcacagattcaaaagttaaattaaaagtatcagcatttaataacaaatagctttgtagagtgcaatcgactgttaaagtggtgtcctgccccagattgccaccatgt tgttaaagtccaatatcctgatgctaaacctgttcgctgcaaatgtgggcgccaattttgctttaactgtggagaaaattggcatgatcctgttaaatgtaagtggt taaagaaatggattaaaaagtgtgatgatgacagtgaaacctccaattggattgcagccaacacaaaggaatgtcccaaatgccatgtcacaattgagaaggatggtggttgtaatcacatggtctgtcgtaaccagaattgtaaagcagagttttgctgggtgtgtcttggcccatgggaaccacatggatctgcctgg tacaactgtaaccgctataatgaggatgatgcaaaggcagcaggctcgagcggtggtggcgggagcggaggtggagggtcgtcaggtgtgaccggctaccggctgttcgaggagattctg

In a preferred embodiment of the present invention, the drug screening method is carried out by As used in the present invention, the term " The "system" refers to a luciferase based on Promega's latest patent The two-subunit system consists of Luciferase is composed of two artificially recombined subunits, namely LgBiT and SmBiT, which can be expressed as fusion proteins with two target proteins to be tested. The two subunits LgBiT and SmBiT have optimal stability and extremely low self-aggregation. The interaction between the target proteins enables the two subunits to bind to obtain an active luciferase, which can then react with the substrate to emit a bioluminescent signal. In an embodiment of the present invention, one of the Ar domain and the Af domain of the ARIH1 protein is expressed as a fusion protein with LgBiT, and the other is expressed as a fusion protein with SmBiT. By utilizing the binding characteristics of the Ar domain and the Af domain of the ARIH1 protein in the self-inhibition state, LgBiT and SmBiT are combined with each other to obtain an active luciferase.

As used in the present invention, the term "Ar domain" refers to ARIH1 protein subunit aa 401-557, and the term "Af domain" refers to ARIH1 protein subunit aa 1-400. The inventors found that the division of ARIH1 protein into Ar subunits and Af subunits is more similar to other divisions (such as aa 185-408 and aa 408-557; aa 185-327 and aa 408-557; aa 1-327 and aa 408-557; aa 105-400 and aa 401-557, etc.). In the system, the stereo configurations of LgBiT and SmBiT are more matched. In the most preferred embodiment of the present invention, the gene sequence encoding the ARIH1 protein subunit (aa 401-557) is inserted into the C-terminus of the LgBit sequence to obtain a first expression vector, i.e., the recombinant plasmid pLgBit-Ar, which can express the fusion protein LgBit-Ar, and the gene sequence encoding the ARIH1 protein subunit (aa 1-400) is inserted into the N-terminus of the SmBit sequence to obtain a second expression vector, i.e., the recombinant plasmid pAf-SmBit, which can fusion express Af-SmBit. The stereo configurations of LgBit-Ar and Af-SmBit match, and the interaction is stronger, which is suitable for use. System development of drug screening methods. In some preferred embodiments of the present invention, the LgBit-Ar fusion protein is shown as SEQ ID NO:3. In some preferred embodiments of the present invention, the Af-SmBit fusion protein is shown as SEQ ID NO:4.

SEQ ID NO:3:

MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITTVTGTLWNGNKIIDERLITPDGSMLFRVTINSGSSGGGGSGGGGSSRDAQERSRAALQRYLFYCNRYMNHMQSLRFEHKLYAQV KQKMEEMQQHNMSWIEVQFLKKAVDVLCQCRATLMYTYVFAFYLKKNNQSIIFENNQADLENATEVLSGYLERDISQDSLQDIKQKVQDKYRYCESRRRVLLQHVHEGYEKDLWEYIED

SEQ ID NO:4:

MDSDEGYNYEFDEDEECSEEDSGAEEEEDEDDDEPDDDTLDLGEVELVEPGLGVGGERDGLLCGETGGGGGSALGPGGGGGGGGGGGGPGHEQEEDYRYEVLTAEQILQHMVECIREVNEVIQNPATITRILLSHFNWDKEKLMERYFDGNLEKLFAECHVINPSKKSRTRQMNTRSSAQDMPCQICYLNYPNSYFTGLECGHKFCMQCWS EYLTTKIMEEGMGQTISCPAHGCDILVDDNTVMRLITDSKVKLKYQHLITNSFVECNRLLKWCPAPDCHHVVKVQYPDAKPVRCKCGRQFCFNCGENWHDPVKCKWLKKWIKKCDDDSETSNWIAANTKECPKCHVTIEKDGGCNHMVCRNQNCKAEFCWVCLGPWEPHGSAWYNCNRYNEDDAKAAGSSGGGGSGGGGSSGVTGYRLFEE IL

The expression of the fusion protein can be achieved by constructing a recombinant plasmid by inserting a polynucleotide sequence encoding the fusion protein into a plasmid vector. In an embodiment of the present invention, the polynucleotide sequence encoding LgBit-Af and the polynucleotide sequence encoding SmBit-Ar are respectively inserted into a plasmid vector to construct recombinant plasmids pLgBit-Af and pSmBit-Ar. In an embodiment of the present invention, the polynucleotide sequence encoding LgBit-Af and the polynucleotide sequence encoding Ar-SmBit are respectively inserted into a plasmid vector to construct recombinant plasmids pLgBit-Af and pAr-SmBit. In an embodiment of the present invention, the polynucleotide sequence encoding LgBit-Ar and the polynucleotide sequence encoding SmBit-Af are respectively inserted into a plasmid vector to construct recombinant plasmids pLgBit-Ar and pSmBit-Af. In an embodiment of the present invention, the polynucleotide sequence encoding SmBit-Af and the polynucleotide sequence encoding Ar-LgBit are respectively inserted into a plasmid vector to construct recombinant plasmids pSmBit-Af and pAr-LgBit. In an embodiment of the present invention, the polynucleotide sequence encoding SmBit-Ar and the polynucleotide sequence encoding Af-LgBit are respectively inserted into a plasmid vector to construct recombinant plasmids pSmBit-Ar and pAf-LgBit. In an embodiment of the present invention, the polynucleotide sequence encoding Af-LgBit and the polynucleotide sequence encoding Ar-SmBit are respectively inserted into a plasmid vector to construct recombinant plasmids pAf-LgBit and pAr-SmBit. In an embodiment of the present invention, the polynucleotide sequence encoding Ar-LgBit and the polynucleotide sequence encoding Af-SmBit are respectively inserted into a plasmid vector to construct recombinant plasmids pAr-LgBit and pAf-SmBit.

In the most preferred embodiment of the present invention, the polynucleotide sequence encoding LgBit-Ar and the polynucleotide sequence encoding Af-SmBit are respectively inserted into plasmid vectors to construct recombinant plasmids pLgBit-Ar and pAf-SmBit. Compared with other recombinant plasmids, the three-dimensional configurations of the fusion proteins expressed by pLgBit-Ar and pAf-SmBit match each other, the interaction force between them is strong, the resulting complex has high enzyme activity, emits a stronger luminescent signal when reacting with the substrate, and has better sensitivity.

In the most preferred embodiment of the present invention, the first coding sequence is a polynucleotide encoding LgBit-Ar fusion protein, and the second coding sequence is a polynucleotide encoding Af-SmBit fusion protein.

The method of transforming, transducing or transfecting a vector containing an exogenous gene into a host cell is a conventional technique in the art. Exemplarily, it can be performed by liposome transfection, calcium phosphate coprecipitation, electroporation transfection or viral infection. In a preferred embodiment of the present invention, the recombinant plasmids pLgBit-Ar and pAf-SmBit are co-transfected into the host cell by liposome transfection. The culture medium (e.g., Opti-mem), transfection reagents (e.g., PEI, etc.) required for liposome transfection, etc. can be obtained commercially. The term "co-transfection" refers to two or more vectors transfecting the same host cell. In an embodiment of the present invention, a mixture of recombinant plasmids pLgBit-Ar and pAf-SmBit is used to co-transfect the host cell, and the ratio of the two is preferably 1:1.

As used in the present invention, the term "vector" refers to a delivery vehicle for polynucleotides. In genetic engineering recombinant technology, a vector includes a polynucleotide sequence encoding a specific protein that can be inserted operably to achieve the expression of the protein. The vector is used to transform, transduce or transfect a host cell, and the genetic material elements delivered by the vector can be expressed in the host cell. The "vector" in the present invention can be any suitable vector, including chromosomes, non-chromosomes and synthetic nucleic acid vectors (including a series of nucleic acid sequences of appropriate expression control elements). For example, the vector can be a recombinant plasmid vector, a recombinant eukaryotic virus vector, a recombinant bacterial phage vector, a recombinant yeast mini-chromosome vector, a recombinant bacterial artificial chromosome vector or a recombinant yeast plasmid vector. In a preferred embodiment of the present invention, the vector is a recombinant plasmid.

As used in the present invention, the term "fusion protein" refers to a molecule that is connected by covalent bonds to two or more proteins or fragments thereof and contained by the main chain of each peptide. Fusion protein is preferably produced by the genetic expression of polynucleotide molecules encoding these proteins. In a preferred embodiment of the present invention, the fusion protein is LgBit-Ar fusion protein or Af-SmBit fusion protein.

As used in the present invention, the term "host cell" refers to a cell into which exogenous polynucleotides and/or vectors are introduced. The host cell is a eukaryotic host cell or a prokaryotic host cell. Wherein, the eukaryotic host cell can be a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algae host cell, a nematode host cell, a protozoan host cell and a fish host cell. Exemplarily, the host cell in the present invention is a eukaryotic host cell, and the eukaryotic host cell is a mammalian host cell. Wherein, the mammalian host cell is a Chinese hamster ovary cell (CHO cell), a COS cell, a Vero cell, a SP2/0 cell, a NS/O myeloid cell, a human fetal kidney cell, an immature hamster kidney cell, a HeLa cell, a human B cell, a cv-1/EBNA cell, a L cell, a 3T3 cell, a HEPG2 cell, a PerC6 cell. In a preferred embodiment of the present invention, the host cell is a 293T cell.

As used in the present invention, the term "luminescent substrate" refers to Any substrate that can generate light through the luciferase reaction of the system is preferably a NanoBit luciferase substrate. The NanoBit luciferase substrate used in the embodiments of the present invention is commercially available.

In a preferred embodiment of the present invention, screening drugs according to changes in the luminescent signal intensity value comprises the step of: screening drugs that reduce the luminescent signal intensity value of host cells or their lysates (preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%) as positive drugs. More preferably, if the luminescent signal intensity value of host cells or their lysates treated with the candidate drug is reduced (preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%) compared to those not treated with the candidate drug, and there is a significant difference (P<0.05), then the drug is a positive drug.

In an embodiment of the present invention, it is feasible to detect the intensity of the luminescent signal of the host cell or its lysate. Any instrument capable of identifying the change in the intensity of the luminescent signal can be used to detect the change in the intensity of the luminescent signal, for example, a fluorescence microscope, an ELISA reader, etc.

In order to facilitate real-time observation, changes in the intensity of the luminescent signal of the host cells themselves can be directly detected.

In order to facilitate high-throughput screening, in a more preferred embodiment of the present invention, the change in the intensity value of the luminescent signal of the host cell lysate is directly detected. Specifically, the host cells are cultured to express Af-SmBit and LgBit-Ar fusion proteins in large quantities, the host cells are lysed, the lysate is treated with the candidate drug, and the change in the intensity value of the luminescent signal is detected.

In a more preferred embodiment, four replicates are set for the candidate drug treatment group and the control group, and a two-tailed t-test is used for testing. If P<0.01, it is considered to be significantly different.

In a preferred embodiment of the present invention, when the candidate drug can inhibit the allosteric activation of ARIH1, that is, it can inhibit the separation of the two subunits Af and Ar of ARIH1, the LgBit fragment and SmBit of the two fusion proteins LgBit-Ar and Af-SmBit are difficult to combine, and the luciferase-catalyzed substrate luminescence cannot be formed, and the luminescent signal intensity of the culture system is correspondingly reduced. This screening method can avoid the differences in cell states caused by possible adverse factors in cell culture, and directly reflect the effect of the drug on the activation of the dual subunit structure of the ARIH1 protein.

Drug Screening System

An embodiment of the present invention also relates to a drug screening system for screening tumor immunotherapy drugs, the drug screening system comprising: a reaction module; the reaction module comprising multiple reaction chambers, the reaction chambers being used to react host cells or their lysates with candidate drugs; a drug delivery module; the drug delivery module being used to deliver candidate drugs to the reaction chambers of the reaction module; a signal recognition module; the signal recognition module being used to identify the luminescent signal intensity value of the reaction chamber; a signal processing module; the signal processing module being used to receive the luminescent signal intensity value from the signal recognition module, and screening positive drugs according to changes in the luminescent signal intensity value.

In a preferred embodiment of the present invention, the reaction chamber includes a culture system containing a luciferase substrate and a culture medium, which is used to culture host cells to express the fusion proteins LgBit-Ar and Af-SmBit. In a preferred embodiment of the present invention, the recombinant vectors pLgBit-Ar and pAf-SmBit are co-transfected into the host cells so that the host cells co-express the fusion proteins LgBit-Ar and Af-SmBit.

In a preferred embodiment of the present invention, the drug delivery module is provided with a connecting portion connecting multiple reaction chambers in the reaction module, and in addition to the candidate drug, the culture fluid for culturing host cells, the luciferase substrate, the host cells or their lysates, etc. can all enter the reaction chamber through the connecting portion. Each reaction chamber should be independent, for example, it can be an independent hole in a microplate, and the size and model of the microplate are not limited, for example, it is a 384-well microplate.

In a preferred embodiment of the present invention, the signal recognition module is provided with a luminescence detection unit, which can identify the luminescence intensity signal, convert it into an electrical signal and transmit the electrical signal to the signal processing module. The signal processing module is provided with a signal amplification module, a data processing and calculation module and a display module to convert the luminescence intensity into graphics or numbers and output them to a display.

In a preferred embodiment of the present invention, the drug screening system also includes a control module for sending a control signal to control the operation of the drug delivery module, the reaction module, the signal recognition module, and the signal processing module. Exemplarily, the control module sends a first signal to the drug delivery module, first the culture fluid including the host cell or its lysate is transported to each reaction chamber through the connecting portion, and then the control module sends a second control signal to the drug delivery module, and the luciferase substrate and the candidate drug are transported to an independent reaction chamber through the connecting portion for reaction. Then the control module sends a third control signal to the signal recognition module, identifies the luminescent signal changes in each reaction chamber, and converts the light signal into an electrical signal. Finally, the control module sends a fourth control signal, transmits the electrical signal to the signal processing module, processes the electrical signal and outputs a visual image or number.

Methods for evaluating anti-tumor immune resistance of drug candidates

An embodiment of the present invention also relates to a method for evaluating the anti-tumor immune resistance of a candidate drug, comprising the steps of: Sa, constructing a host cell, the host cell being capable of expressing LgBit-Ar fusion protein and Af-SmBit fusion protein; Sb, culturing the host cell in a culture system, treating the host cell or its lysate with a candidate drug, adding a luminescent substrate, detecting and recording the change in the luminescence intensity value of the host cell or its lysate; Sc, when the luminescence intensity value of the host cell or its lysate treated with the candidate drug is reduced (preferably reduced by at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%) compared with a control group not treated with the candidate drug, and there is a significant difference (P<0.05), it is determined that the candidate drug has anti-tumor immune resistance.

In a preferred embodiment, an enzyme-linked immunosorbent assay (ELISA) is used to measure the luminescence intensity value of the culture system.

drug

The present invention also relates to drugs obtained by screening the drug screening method or drug screening system of the present invention. The drug of the present invention has the effect of promoting the allosteric activation of ARIH1, can promote the ubiquitination activity of ARIH1 protein, can effectively alleviate ICB resistance, and can be used as a tumor immunotherapy drug or adjuvant therapy drug.

To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and/or".

As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

Example 1: Establishment of intracellular ARIH1 allosteric activation screening model

In this example, NanoBit technology is used to construct a high-throughput screening method for activation of ARIH1 allosteric subunits in living cells. ARIH1 protein has structural characteristics of regulatory domains and functional domains. In the silent state, its regulatory domain and functional domain are tightly connected. When the protein function is activated, the tight connection between the subunits will dissociate. This characteristic is combined with NanoBit technology to achieve high-throughput screening of small molecule drugs targeting ARIH1.

The working principle of NanoBit technology is to split the NanoBit protein into 17.6kDa LgBit and 11 amino acids SmBit, and use them as two tags on the target protein expression vector. When there is interaction between the two target proteins, the two subunits of NanoBit will undergo structural complementation to form a complete NanoBit protein, which will emit a strong luminescent signal under substrate detection. The specific screening principle is shown in Figure 1.

S1: First, using the LgBit and SmBit vectors provided by NanoBiT ^TM PPI MCS Starter System (promega), eight detection vectors were constructed according to the N-terminus and C-terminus of the amino acid sequences of LgBit and SmBit fused to the Af protein region (aa 1-400) and Ar protein region (aa 401-557) of ARIH1. In order to ensure that the target proteins obtained by separately expressing the ARIH1 protein subunits can interact stably with each other, experiments were conducted according to eight combinations of pLgBit-Af and pSmBit-Ar, pLgBit-Af and pAr-SmBit, pLgBit-Ar and pSmBit-Af, pLgBit-Ar and pAf-SmBit, pSmBit-Af and pAr-LgBit, pSmBit-Ar and pAf-LgBit, pAf-LgBit and pAr-SmBit, and pAr-LgBit and pAf-SmBit (Figure 2). The plasmids of the above combination were co-transfected into 293T cells at a molecular ratio of 1:1. After 48 hours of transfection, a final concentration of 10 μM luminescent substrate was added and detected using an ELISA instrument. The test results are shown in Figure 3. The luminescence intensity detection signal of the interaction of the fusion protein expressed by the pLgBit-Ar and pAf-SmBit plasmid combination is stronger, indicating that it simulates the strong interaction between the regulatory structure region Ar and the functional structure region Af under the autoinhibitory state in the cell. In order to verify the allosteric activation characteristics of the pLgBit-Ar and pAf-SmBit plasmid combination, point mutations were made to the Ar domain to construct pLgBit-Ar-OpenA (F430/E431/E503) and pLgBit-Ar-OpenB (R420/N423/E503) constitutively activated mutants. The above two mutants were previously reported in the literature to have a weakened ability of the Ar region to shield the catalytic active region Af, so ARIH1 has constitutively activated characteristics. The test results are shown in Figure 4. The luminescence value of the combination was significantly reduced after mutation, proving that the pLgBit-Ar and pAf-SmBit plasmid combination reflects allosteric activation characteristics and is suitable for subsequent high-throughput screening of small molecule drugs.

S2: After determining that pLgBit-Ar and pAf-SmBit are the optimal screening combinations, 293T cells were cultured in a 90mm culture dish at a cell density of 2x10 ⁶ , and the culture medium was DMEM (VivaCell) containing 10% serum and 1% double antibody. When the cell confluence reached 90%, the two recombinant plasmids were mixed according to the transfection system of PEI 12μL, plasmid 6μg, and Opti-mem 500μL, and allowed to stand at room temperature for 20 minutes. The recombinant plasmids were transfected into 293T cells by co-transfection with liposomes for 12 hours, so that 293T cells can stably express the target protein.

S3: Observe the cell growth status, use 1ml of trypsin to suspend the well-growing cells and collect them into a centrifuge tube, and centrifuge at 1000r for 3 minutes to collect the cell precipitate. Use 10ml of cell culture medium to blow and mix the cells, and use a cell counter (MONWEI) to count the cells. Dilute the cells according to the number of cells corresponding to 1x10 ⁵ per 1ml of cell suspension, and transfer the cells to a 384-well plate at 50μL per well. When the cell confluence reaches more than 95%, add the luminescent substrate (working concentration 10μM) to react, and detect the change in the luminescence value of the cells in the microplate reader. On this basis, small molecule drugs are added and screened on a 384-well plate. The changes in luminescence values corresponding to different small molecule drugs detected are compared with the components without small molecule drugs added, and the decrease in the luminescence value corresponding to each drug is calculated. According to the decrease in the luminescence value of different small molecule drugs, drugs with potential as tumor immunotherapy drugs are screened. FIG5 is a test of the effect of a positive drug (Compound) on cells. The results indicate that the drug promotes the allosteric activation of ARIH1, and this effect increases with increasing drug concentration.

Example 2: Establishment of an in vitro ARIH1 allosteric activation luminescence detection model

This study used NanoBit technology to establish a high-throughput screening method for extracellular ARIH1 allosteric subunit activation.

S1: 293T cells were cultured in a 90mm culture dish. When the 293T cells grew to 90% confluence, pLgBit-Ar plasmid and pAf-SmBit plasmid were transfected into 293T cells according to the transfection system of PEI 12μL, plasmid 6μg, and Opti-mem 500μL, and cultured for 24h. The cells were digested with 1ml of trypsin and collected into a centrifuge tube. The cell pellet was collected by centrifugation and the cells were washed twice with 10ml of PBS. The cells were lysed by adding a cell lysis buffer without detergent, and the cells were treated by repeated freezing and thawing and low-power ultrasonic disruption. The target protein required for the experiment was obtained by centrifugation.

S2: Add equal amounts of pLgBit-Ar and pAf-SmBit proteins to a centrifuge tube, mix well, and react at 4°C for 1 hour. Add 8 μL of the protein mixture to a 384-well microplate, add 2 μL of small molecule drugs to each well, and react at 4°C. Then add 10 μL of luminescent substrate (working concentration 10 μM) and detect the luminescent signal in an ELISA reader.

In the example, the in vitro allosteric activation of ARIH1 protein by small molecule drugs at different concentrations was detected. As shown in FIG6 , the luminescence value decreases with the increase of the concentration of the positive drug (Compound), that is, the effect of the positive drug on the allosteric activation of ARIH1 increases with the increase of the drug concentration.

Example 3: Thermal Shift to detect the effect of small molecule drugs on the thermal stability of ARIH1

This experiment uses thermal-shift assay technology to detect the interaction between small molecule drugs and ARIH1 protein, and determines the small molecule drugs targeting ARIH1 protein by measuring the changes in protein stability at different heating temperatures.

S1: Add 1 μg of highly purified ARIH1 protein to each well of a 96-well qPCR plate. Orange protein dye and 2 μL of small molecule drugs were added to the reaction system to make up to 20 μL with PBS, and then allowed to stand at 4°C for 30 minutes.

S2: Transfer the qPCR plate to the qPCR instrument to detect changes in the protein dissolution curve, use Protein ThermalShift Software to analyze the obtained detection data, and screen drugs that bind to the ARIH1 protein based on changes in protein Tm treated with different small molecule drugs and changes in the peak type of the protein dissolution curve.

In this example, the inventors tested the effect of drugs on the thermal stability of ARIH1. As shown in FIG7 , the ΔTm of the ARIH1 protein changed significantly after the addition of the positive drug (Compound), proving that the drug interacts with the ARIH1 protein at the biochemical level.

Example 4: Detection of the effect of drugs on ARIH1 ubiquitin ligation activity

S1: 293T cells were cultured in a 100 mm culture dish and grown to 90% confluence. ARIH1 ^-WT and ARIH1 ^-C357S protein expression plasmids were transfected into the cells by liposome transfection and cultured for 48 hours.

S2: ARIH1 ^-WT protein and ARIH1 ^-C357S enzyme-inactive mutant were purified using HA-tagged protein immunoprecipitation kit.

S3: Add each reaction component according to the following reaction system and set up a control group and a test group, and react at 37°C for 30 minutes. After the reaction, add 5 μL of 5xSDS-protein loading buffer and heat the sample at 100°C for 10 minutes.

S4: Western Blot test was performed on the samples to detect the Ub protein content and its modification changes in the samples. As shown in Figure 8, when different concentrations of positive drugs (Cp) were added, the ubiquitination level of the wild-type ARIH1 ^-WT protein system increased significantly at a positive drug (Cp) concentration of 10μM; while the ubiquitination of the enzyme-inactive mutant ARIH1 ^-C357S system did not change significantly, indicating that the added positive drug can promote the ubiquitination activity of the ARIH1 protein and has the potential to be used as a drug targeting the ARIH1 protein and tumor immunity.

Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims (10)

1. A method of screening for a drug for use in tumor immunotherapy, the method comprising the steps of:

s1, respectively constructing a first expression vector containing a first coding sequence and a second expression vector containing a second coding sequence, wherein the first coding sequence is shown as SEQ ID NO. 1; the second coding sequence is shown as SEQ ID NO. 2 code;

S2, co-transfecting the first expression vector and the second expression vector into a host cell and culturing the host cell;

S3, treating the host cells or lysate thereof with a candidate drug; and

S4, adding a luminescent substrate, detecting the luminous signal intensity of the host cell or the lysate thereof, and screening the medicine according to the change of the luminous signal intensity value.

2. The method according to claim 1, wherein the method is carried out byThe system screens candidate drugs.

3. The method of claim 2, wherein the host cell or lysate thereof is screened for a drug that reduces the luminescent signal intensity value.

4. The method of claim 3, wherein the drug is a positive drug if the host cell or lysate thereof treated with the candidate drug has a reduced luminescence signal intensity value and a significant difference (P < 0.05) compared to the host cell not treated with the candidate drug.

5. The method of claim 2, wherein the first expression vector and the second expression vector are co-transfected into the host cell in a 1:1 ratio.

6. The method of claim 2, wherein the host cell is a 293T cell.

7. A drug screening system, the drug screening system comprising:

a reaction module; the reaction module comprises a plurality of reaction cavities, wherein the reaction cavities contain host cells for screening medicines and a culture system thereof;

A drug delivery module; the drug delivery module is used for delivering a candidate drug into a reaction cavity of the reaction module;

A signal identification module; the signal identification module is used for identifying the luminous signal intensity value of the reaction cavity; and

A signal processing module; the signal processing module is used for receiving the luminous signal intensity value from the signal identification module and screening positive medicines according to the luminous signal intensity value change;

Wherein the host cell is capable of co-expressing LgBit-Ar fusion protein and Af-SmBit fusion protein.

8. The drug screening system of claim 7, wherein the LgBit-Ar fusion protein is set forth in SEQ ID No. 3; and/or the Af-SmBit fusion protein is shown as SEQ ID NO. 4.

9. The drug screening system of claim 7, wherein the host cell comprises the first expression vector and the second expression vector.

10. The method according to any one of claims 1-6 or the drug screening system according to any one of claims 7-9, screening the resulting drug.