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WO2024011360A1 - Système de criblage basé sur le stress de survie et dispositif de criblage à haut débit - Google Patents

Système de criblage basé sur le stress de survie et dispositif de criblage à haut débit Download PDF

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WO2024011360A1
WO2024011360A1 PCT/CN2022/104945 CN2022104945W WO2024011360A1 WO 2024011360 A1 WO2024011360 A1 WO 2024011360A1 CN 2022104945 W CN2022104945 W CN 2022104945W WO 2024011360 A1 WO2024011360 A1 WO 2024011360A1
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protein
fusion protein
trp1p
drug
fusion
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PCT/CN2022/104945
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Chinese (zh)
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刘翔宇
何国栋
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清华大学
北京宏岭隆成科技有限公司
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Priority to PCT/CN2022/104945 priority Critical patent/WO2024011360A1/fr
Publication of WO2024011360A1 publication Critical patent/WO2024011360A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase

Definitions

  • the present disclosure belongs to the field of biomedical technology. Specifically, the present disclosure relates to a fusion protein composition, a fusion protein, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant yeast strain, a survival pressure-based screening system, and a high-throughput screening device. and uses, as well as methods for constructing recombinant yeast strains and methods for drug screening.
  • GPCR G Protein Coupled Receptor
  • GPCRs are the largest membrane protein receptor superfamily known in eukaryotic organisms, with more than 800 subtypes in the human genome 1 .
  • GPCRs are widely distributed on the surface of various types of cells in the human body. They can sense a variety of extracellular signaling molecules, such as hormones, neurotransmitters, peptides and lipids, and transmit extracellular signals to cells by activating their downstream signal transduction pathways. Inside. Because it is widely distributed on the cell surface and has very important physiological functions, GPCR is currently one of the most important drug targets. According to statistics, among all clinical drugs approved by the FDA, more than 33% of them target various GPCRs. GPCR drugs include their agonists or antagonists, which have a therapeutic effect on diseases by activating or inhibiting the functions of various GPCRs2.
  • GPCR ligands The important medicinal value of GPCR ligands has always made them favored by the pharmaceutical industry.
  • drug screening methods for GPCRs have been developed, mainly divided into screening methods based on ligand-receptor interactions (for example, based on surface plasmon resonance technology) 3 , screening methods based on the biological function of the receptor (for example, by indirectly detecting secondary messengers such as calcium ions or cAMP downstream of intracellular receptors) 4 and structure-based screening methods (such as virtual screening) ) 5 .
  • Each of these methods has its own pros and cons.
  • function-based screening methods are often the most direct and effective.
  • Candidate compounds obtained from interaction or structure-based screening methods need to undergo biological functional verification before their activity can be confirmed.
  • DMSO solvent dimethyl sulfoxide
  • these function-based screening methods are often insensitive to relatively weak candidate compounds, resulting in missing some important candidate compounds, especially those with novel skeletons.
  • existing function-based screening methods usually detect receptor-triggered intracellular signals such as calcium ions and cAMP. Due to the complex biological metabolism network in cells, these responses are usually non-orthogonal. Small molecule compounds may show false positive results by inducing responses in other signaling pathways, causing greater interference to the normal screening process. .
  • An object of the present disclosure is to provide a fusion protein that can be used to construct a survival pressure selection system for drug screening.
  • Another object of the present disclosure is to provide a composition of a fusion protein that directly feeds back the activation or inhibition of GPCR as a survival reporter signal.
  • Another object of the present disclosure is to provide a simple and stable recombinant yeast cell or survival pressure-based screening system that can be used for GPCR drug screening.
  • Another object of the present disclosure is to provide a high-throughput screening device capable of high-throughput screening of GPCR drugs.
  • Another object of the present disclosure is to provide a drug screening method that can improve the sensitivity and accuracy of GPCR drug screening.
  • a fusion protein composition wherein the composition includes the fusion protein I shown in the following (i) and the fusion protein II shown in the following (iv) that exist independently; or, the combination
  • the substance includes the fusion protein I shown in the following (ii) and the fusion protein II shown in the following (iii) that exist independently:
  • the first protein is selected from G protein-coupled receptors or functional variants thereof
  • the second protein is selected from G proteins or functional variants thereof
  • the first domain is selected from the carboxyl terminal structure of a reporter protein Domain
  • the second domain is selected from the amino-terminal domain of the reporter protein
  • the carboxyl-terminal domain of the reporter protein and the amino-terminal domain of the reporter protein are used to form a reporter protein, and the reporter protein releases a reporter signal for detection.
  • the reporter protein is selected from a key enzyme in the metabolic pathway encoded by an auxotrophic screening marker gene; preferably, the auxotrophic screening marker gene is a gene encoding phosphoribosyl anthranilate isomerase (trp1 ).
  • the auxotrophic screening marker gene is a gene encoding phosphoribosyl anthranilate isomerase (trp1 ).
  • [3] The fusion protein composition according to [1] or [2], wherein the reporter protein is phosphoribosyl anthranilate isomerase (Trp1p), and the first domain is phosphoribosyl anthranilate isomerase (Trp1p).
  • the second domain is the amino-terminal domain of phosphoribosyl anthranilate isomerase (Trp1p NTD );
  • the first structural domain is selected from any one of the following (a 1 )-(a 2 ):
  • polypeptide that has at least 80% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 3, and has or partially has the sequence activity shown in SEQ ID NO: 3;
  • the second domain is selected from any one of the following (b 1 )-(b 2 ):
  • fusion protein composition according to any one of [1] to [3], wherein the first protein is selected from any one of the following: ⁇ 2 -adrenergic receptor ( ⁇ 2 AR) or Its functional variant, human melanocortin receptor-4 (MC4R) or its functional variant, melanocortin receptor 1 (MC1R) or its functional variant;
  • ⁇ 2 AR ⁇ 2 -adrenergic receptor
  • M4R human melanocortin receptor-4
  • M1R melanocortin receptor 1
  • the first protein is carboxyl-terminal truncated ⁇ 2-adrenoceptor ( ⁇ 2AR ), human melanocortin receptor-4 (MC4R) or melanocortin receptor 1 (MC1R);
  • ⁇ 2AR carboxyl-terminal truncated ⁇ 2-adrenoceptor
  • M4R human melanocortin receptor-4
  • M1R melanocortin receptor 1
  • the first protein is selected from any one of the following (c 1 )-(c 2 ):
  • the fusion protein I is formed by fusing the first structural domain to the interior of the first protein
  • the fusion protein I is formed by fusing the second structural domain to the carboxyl terminus of the first protein;
  • the fusion protein I is formed by fusing the second structural domain to the interior of the first protein
  • the fusion protein I includes a connecting peptide connecting the first structural domain and the first protein; or, the fusion protein I includes a connecting peptide connecting the second structural domain and the first protein. peptide;
  • the fusion protein I has a structure shown in any one of the following (j 1 ) to (j 16 ):
  • the fusion protein I is selected from any one of the following (e 1 )-(e 2 ):
  • the fusion protein II includes a second connecting peptide connecting the first structural domain and the second protein; or, the fusion protein II includes a second connecting peptide connecting the second structural domain and the second protein.
  • the fusion protein II has a structure shown in any one of the following (k 1 ) to (k 4 ):
  • the miniG protein is formed by the fusion of the miniG protein fragment I and the miniG protein fragment II;
  • the fusion protein II is selected from any one of the following (f 1 )-(f 2 ):
  • (f 2 ) has at least 80% sequence identity with the amino acid sequence described in any one of SEQ ID NO: 14-17, and has or partially has as shown in any one of SEQ ID NO: 14-17 Sequentially active proteins.
  • a fusion protein selected from any one of the following groups (i)-(iv):
  • the first protein is selected from G protein-coupled receptors or functional variants thereof
  • the second protein is selected from G proteins or functional variants thereof
  • the first domain is selected from the carboxyl terminal structure of a reporter protein Domain
  • the second domain is selected from the amino-terminal domain of the reporter protein
  • the carboxyl-terminal domain of the reporter protein and the amino-terminal domain of the reporter protein are used to form a reporter protein, and the reporter protein releases a reporter signal for detection.
  • a recombinant nucleic acid molecule wherein the recombinant nucleic acid molecule comprises a nucleotide sequence encoding the composition of the fusion protein according to any one of [1] to [7].
  • a first coding region comprising a first coding sequence encoding fusion protein I
  • the second coding region comprising a second coding sequence for encoding fusion protein II;
  • the first coding sequence is selected from any one of the following (g 1 )-(g 2 ):
  • (g 2 ) has at least 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 18-21, and encodes a sequence shown in any one of SEQ ID NO: 18-21 The nucleotide sequence of the protein encoding protein activity;
  • the second coding sequence is selected from any one of the following (h 1 )-(h 2 ):
  • (h 2 ) has at least 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 22-25, and encodes a sequence shown in any one of SEQ ID NO: 22-25 The nucleotide sequence encoding a protein with protein activity.
  • the promoter is a bidirectional promoter connecting the first coding region and the second coding region;
  • the first coding region is located upstream of the bidirectional promoter, and the second coding region is located downstream of the bidirectional promoter; preferably, the bidirectional promoter is GAL1,10.
  • a recombinant expression vector wherein the recombinant expression vector contains the recombinant nucleic acid molecule according to any one of [10]-[12].
  • composition of the fusion protein according to any one of [1] to [7], the fusion protein according to [8], the polynucleotide according to [9], the composition according to [10]-[ 12] The use of the recombinant nucleic acid molecule according to any one of the above, or the recombinant expression vector according to [13] in at least one of the following (1)-(2):
  • the drug is a GPCR drug, more preferably a GPCR agonist.
  • a recombinant yeast cell wherein the recombinant yeast cell expresses the fusion protein composition as described in any one of [1]-[7]; or, the recombinant yeast cell contains the fusion protein as described in [10]- [12]
  • the recombinant yeast cell is derived from Saccharomyces cerevisiae, more preferably tryptophan auxotrophic Saccharomyces cerevisiae, more preferably trp1 gene deficient Saccharomyces cerevisiae.
  • [16] A method for constructing recombinant yeast cells, wherein the method includes using the recombinant nucleic acid molecule as described in any one of [10]-[12], or the recombinant expression vector as described in [13] Steps to transfer into yeast cells;
  • the yeast cell is a Saccharomyces cerevisiae cell, more preferably a tryptophan auxotrophic Saccharomyces cerevisiae cell, and more preferably a trp1 gene-deficient Saccharomyces cerevisiae cell.
  • a screening system based on survival pressure wherein the screening system includes a fusion protein composition as described in any one of [1]-[7], such as any one of [10]-[12]
  • the recombinant nucleic acid molecule is a recombinant expression vector as described in [13], or a recombinant yeast cell as described in [15].
  • a high-throughput screening device wherein the high-throughput screening device includes:
  • Drug module used to store drugs to be screened
  • a strain inoculation module used to inoculate the recombinant yeast cells as described in [15] into the drug module, so that the recombinant yeast cells come into contact with the drug to be screened;
  • a culture module used for cultivating the recombinant yeast cells inoculated in the drug module
  • a measurement module used to obtain the growth rate of the recombinant yeast cells cultured in the culture module
  • a determination module used to determine whether the drug to be screened is a target screening drug based on the growth rate of the recombinant yeast cells
  • the high-throughput screening system further includes:
  • a transfer module is used to transfer the drug to be screened to the drug module.
  • the drug module includes a multi-well plate with an array of micro-wells, the micro-wells are independently arranged on the multi-well plate, and the drugs to be screened are stored in a first preset number of the micro-wells;
  • the strain inoculation module is used to inoculate the recombinant yeast cells into the first preset number of microwells;
  • the first preset number is ⁇ 50, preferably ⁇ 100, and more preferably ⁇ 300.
  • the measurement module is used to obtain the OD value of the recombinant yeast cells cultured in the culture module;
  • the determination module is used to determine whether the drug to be screened is a target screening drug according to the OD value of the recombinant yeast cell;
  • the target screening drug is selected from GPCR agonists, GPCR antagonists or GPCR reverse inhibitors.
  • a method of drug screening wherein the method includes:
  • Drug treatment step use the drug to be screened to treat the recombinant yeast cells
  • Comparing step Compare the growth rate of the recombinant yeast cells treated with the drug with the growth rate of the recombinant yeast cells not treated with the drug;
  • Determining step judging whether the drug to be screened is a target screening drug based on the growth rate of the recombinant yeast cells;
  • the screening medium is a medium for screening GPCR agonists
  • the medium for screening GPCR agonists is selected from an auxotrophic medium, or a culture containing a drug for inhibiting yeast growth. base;
  • the screening medium is a medium used for screening GPCR antagonists or reverse inhibitors
  • the medium used for screening GPCR antagonists or reverse inhibitors includes: auxotrophic medium and GPCR agonist
  • the target screening drug is selected from GPCR agonists, GPCR antagonists or GPCR reverse inhibitors.
  • the fusion protein provided by the present disclosure can be used to construct a survival pressure screening system for drug screening in cells by directly feeding back the reporter signal generated after the GPCR is activated, or by feeding back the activated GPCR to be activated due to the activation effect.
  • the reporting signal generated by the weakening or elimination of competition improves the sensitivity and reliability of GPCR drug screening.
  • the fusion protein composition provided by the present disclosure includes fusion protein I and fusion protein II that exist independently. Fusion protein I and fusion protein II are formed by splitting the reporter protein into two domains and fusion with GPCR and G protein respectively. When the GPCR is activated, fusion protein I and fusion protein II combine with each other, causing the first domain and the second domain of the reporter protein to approach each other and combine to form a functionally active reporter protein, and feedback a detectable survival reporter signal.
  • the fusion protein I and fusion protein II designed based on protein fragment complementation in this disclosure can directly feed back the survival reporting signal generated by the combination of GPCR and G protein without going through some transcriptional regulation and cascade amplification processes, making the signal more real and reliable. It has the advantages of high sensitivity, less interference from non-orthogonal signal responses, and high reliability.
  • the recombinant yeast cells provided by the present disclosure establish a signal response system orthogonal to the human GPCR-G protein signaling pathway by expressing fusion protein I and fusion protein II in yeast cells.
  • Recombinant yeast cells can convert the activation process of GPCR into the growth process of yeast, or convert the process in which the activated GPCR is competitively weakened or eliminated due to the activation effect into the growth inhibition process of yeast, producing highly sensitive, direct and reliable growth reports.
  • Signal suitable for screening drug molecules with low affinity to GPCR or weak activation effect, and has great application prospects in clinical drug screening.
  • the GPCR-G protein signaling pathway constructed in yeast cells in the present disclosure does not interact with the yeast's own signal transduction pathway, and false positive results caused by signal cross-linking responses can be avoided to a large extent.
  • the final feedback signal of the survival reporter protein from the recombinant yeast cells only comes from the activation or inhibition of the target GPCR and will not be interfered by other factors, avoiding the possible stress response of the yeast itself under growth pressure on the reporter system. influence, the system is more stable and reliable.
  • the method for constructing recombinant yeast cells provided by the present disclosure does not require complex transformation or gene editing of yeast strains, and has the advantages of simple steps and easy implementation.
  • the recombinant yeast cells provided by the present disclosure are constructed based on fusion protein I and fusion protein II obtained by splitting the reporter protein encoded by the auxotrophic screening marker gene and fusing it with GPCR and G protein.
  • the GPCR in fusion protein I is activated by combining with the drug to be screened, it binds to the G protein in fusion protein II, bringing the two domains of the reporter protein close to each other, and combines to form a functionally active reporter protein, making the recombinant yeast cells
  • the growth rate is significantly improved, enabling high sensitivity, high specificity and large-scale screening of drug molecules to be screened.
  • the present disclosure provides recombinant yeast cells grown in auxotrophic media containing GPCR agonists.
  • the GPCR in fusion protein I is activated after binding to a low concentration of GPCR agonist, and binds to the G protein in fusion protein II, bringing the two domains of the reporter protein close to each other, and combine to form a functionally active reporter protein for Maintain growth of recombinant yeast cells in auxotrophic media.
  • the drug to be screened to the culture medium, if the growth rate of the recombinant yeast cells slows down or stops, it means that the drug to be screened is an antagonist or reverse inhibitor that can competitively bind to the GPCR in fusion protein I with the GPCR agonist. agent to achieve high sensitivity, high specificity and large-scale screening of GPCR antagonists or GPCR reverse inhibitors.
  • the recombinant yeast cells provided by the present disclosure are constructed by using the resistance protein as the reporter protein, splitting the resistance protein and fusing it with GPCR and G protein to obtain fusion protein I and fusion protein II.
  • the GPCR in fusion protein I is activated by combining with the drug to be screened, it binds to the G protein in fusion protein II, bringing the two domains of the reporter protein close to each other, and combines to form a functionally active reporter protein, so that the recombinant yeast cells can Grow in a medium containing growth inhibitory drugs to achieve high sensitivity, high specificity and large-scale screening of drug molecules to be screened.
  • the high-throughput screening device provided by the present disclosure can realize large-scale, high-throughput screening of GPCR drugs, especially GPCR drugs with relatively weak affinity to GPCR, and is of great importance in clinical drug screening. Value.
  • the survival pressure-based screening system provided by the present disclosure has the following advantages:
  • this disclosure Based on the structural information of GPCR, G protein and reporter protein, this disclosure accurately and intuitively designs the formation of fusion protein I and fusion protein II based on protein fragment complementation, and innovatively splits the reporter protein into two parts. Each part is fused to the target GPCR and G protein.
  • the survival reporting signal fed back by the reporter protein comes directly from the mutual binding of the receptor and the G protein without going through a series of transcriptional regulation and cascade amplification, making the signal more authentic and credible.
  • This disclosure establishes a signal response system in yeast cells that is orthogonal to the human GPCR-G protein signaling pathway.
  • the activation of the target GPCR does not have any interaction with the yeast's own signal transduction pathway, except for the yeast's own color.
  • Intermediates related to the amino acid anabolism pathway may have an impact.
  • the final reporter protein signal only comes from the activation of the target GPCR without interference from other factors, avoiding the possible stress that may occur in the yeast itself under growth pressure. The impact of stimulus reactions on the reporting system is reduced, making the system more stable and reliable.
  • Figure 1 shows the principle and design schematic diagram of the Survival Pressure Selection (SPS)-based screening system in the present disclosure
  • SPS Survival Pressure Selection
  • a. is a model schematic diagram of the SPS system.
  • b. Schematic diagram of protein structure design in SPS system.
  • Figure 2 shows the strain growth rate of EBY100- ⁇ 2AR -SPS in the present disclosure under different ⁇ 2AR agonist or antagonist treatments.
  • BI-167107 is a high-affinity ⁇ 2 AR complete agonist
  • alprenolol is a ⁇ 2 AR antagonist
  • Apo is a control without GPCR ligand added.
  • Figure 3 shows the results of the agonist-dependent assay of EBY100- ⁇ 2 AR-SPS.
  • a. is the strain growth curve of EBY100- ⁇ 2 AR-SPS treated with ⁇ 2 AR agonist isoproterenol and epinephrine; OD 600 is measured using a microplate reader, and the results are normalized according to isoproterenol. After chemical processing, the data are presented as the mean ⁇ standard error, with 6 replicates in each group.
  • b Schematic diagram of the agonist-dependent testing process of the SPS system; after the first round of screening of EBY100- ⁇ 2 AR-SPS yeast, the yeast grown under agonist stimulation will be treated with tryptophan containing only L-ascorbic acid.
  • the defective culture medium was diluted and divided into three parts, respectively supplemented with the same concentration of agonist, antagonist or no ligand for the second round of screening. Place the newly diluted yeast into a 50 ml sterile tube and culture it with shaking at 25 degrees Celsius and 250 rpm, and observe its growth.
  • cd. shows the growth detection results of EBY100- ⁇ 2 AR-SPS after two rounds of screening; OD 600 was measured using a spectrophotometer, and the difference from the initial OD 600 was calculated.
  • Figure 4 shows the growth test results of EBY100-MC4R-SPS in this disclosure.
  • Dimethyl sulfoxide was added to the control group, and SET was the exogenous agonist Setmelanotide.
  • Figure 5 shows a high-throughput screening platform for GPCR agonists based on the SPS system.
  • a Schematic flow chart of the high-throughput screening platform for GPCR agonists based on the SPS system.
  • b MC4R agonist screening results.
  • c Biochemical functional verification of chlorprothixol.
  • Figure 6 shows the miniG protein sequence, a schematic diagram of the miniG protein secondary structure, and multiple fusion positions (site 1, site 2, and site 3) existing in the miniG protein.
  • Figure 7 shows the results of growth testing of EBY100-MC1R-SPS yeast in the present disclosure.
  • Figure 8 shows a schematic diagram of the secondary structure of the interaction between fusion protein I and fusion protein II in the SPS system.
  • Figure 9 shows the results of growth testing of EBY100-SPS-MC4R-p2 yeast in the present disclosure.
  • polypeptide As used in this disclosure, the terms "polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • Polypeptides can be isolated from natural sources, can be produced by recombinant techniques from eukaryotic or prokaryotic hosts and can be the product of synthetic methods.
  • fusion protein refers to a polypeptide, protein having peptide sequences that are not linked in nature.
  • the fusion protein can be a target protein obtained by the fusion of two or more proteins in prokaryotic or eukaryotic cells using genetic engineering technology; it can also be a target protein obtained by using synthetic technology and fused with two or more polypeptides. protein.
  • auxotroph means that an organism or cell must be cultured in a medium containing essential nutrients that the auxotrophic organism cannot synthesize by itself.
  • auxotrophic markers include pyrimidines, tryptophan, adenine, inositol, leucine, and the like.
  • the auxotroph is a tryptophan auxotroph.
  • auxotrophic selection marker gene refers to a marker sequence that encodes a gene product capable of compensating for an auxotrophy of an organism or cell, thereby rendering the auxotrophic organism or cell prototrophic.
  • the gene product of the auxotrophic selection marker gene promotes the synthesis of such deficient essential nutrients by auxotrophic cells. Therefore, expression of the auxotrophic selection marker gene eliminates the need to add this essential nutrient to the culture medium in which the organism or cell is cultured because the organism or cell has already acquired prototrophy.
  • the protein encoded by the auxotrophic selection marker gene is also called a reporter protein.
  • GPCR G Protein Coupled Receptor
  • GPCR G Protein Coupled Receptor
  • the number and type of the receptors on the cell surface are The most in the middle. GPCRs are widely present in organisms ranging from lower fungi to higher mammals.
  • a GPCR of the present disclosure is a G protein-coupled receptor.
  • G protein-coupled receptors include, but are not limited to, ⁇ 2 - adrenergic receptor ( ⁇ 2 AR), human melanocortin receptor-4 (melanocortin 4receptor, MC4R), melanocortin receptor Melanocortin 1receptor (MC1R), human adenosine A2A receptor, chemokine receptors CXCR4, CCR5, etc.
  • the G protein-coupled receptor or functional variant thereof is selected from wild-type GPCR, GPCR functional variant.
  • Functional variants of GPCRs include, but are not limited to, mutants of wild-type GPCRs, modified GPCRs, GPCR protein fragments, genetically engineered GPCRs, etc.
  • GPCR functional variants may have increased or decreased protein activity of the G protein-coupled receptor.
  • a GPCR functional variant has a GPCR of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher protein activity.
  • ⁇ 2 -adrenoceptor or functional variant thereof is selected from wild-type ⁇ 2AR , ⁇ 2AR functional variant.
  • Functional variants of ⁇ 2 AR include, but are not limited to, mutants of wild-type ⁇ 2 AR, modified ⁇ 2 AR, ⁇ 2 AR protein fragments, genetically engineered ⁇ 2 AR, etc.
  • Functional variants of ⁇ 2AR may have increased or reduced protein activity of ⁇ 2AR .
  • the ⁇ 2AR functional variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher ⁇ 2 AR protein activity.
  • human melanocortical receptor-4 or functional variants thereof are selected from wild-type MC4R, MC4R functional variants.
  • Functional variants of MC4R include, but are not limited to, mutants of wild-type MC4R, modified MC4R, MC4R protein fragments, genetically engineered MC4R, etc.
  • MC4R functional variants may have increased or decreased protein activity of MC4R.
  • the MC4R functional variant has an MC4R of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher protein activity.
  • G protein refers to a class of signal transduction proteins that can bind to guanine nucleotides and have GTP hydrolase activity. GPCR regulates the activity of related intracellular enzymes by binding to G proteins, thereby generating second messengers to transmit ligand signals from extracellular transmembranes to intracellular, changing the functional activity of cells and ultimately affecting the growth of organisms. Development, reproduction, diapause, feeding, metabolism and behavior play a regulatory role.
  • G protein or functional variant thereof is selected from wild-type G protein, G protein functional variant.
  • Functional variants of G protein include, but are not limited to, mutants of wild-type G protein, modified G protein, G protein protein fragments, genetically engineered G protein, etc.
  • G protein functional variants may have increased or decreased protein activity of the G protein.
  • the G protein functional variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher G protein protein activity.
  • wild-type refers to a subject that can be found in nature.
  • a polypeptide, polynucleotide sequence, or microorganism that can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring.
  • naturally occurring and “wild type” are synonymous.
  • mutant refers to a polynucleotide or polypeptide that contains an alteration at one or more (e.g., several) positions (i.e., several) relative to a "wild-type", or “comparison” , substituted, inserted and/or deleted polynucleotides), where substitution refers to the replacement of a nucleotide occupying a position with a different nucleotide.
  • Deletion refers to the removal of a nucleotide occupying a certain position.
  • Insertion refers to the addition of a nucleotide adjacent to and immediately following the nucleotide occupying the position.
  • polynucleotide and “nucleic acid molecule” refer to a polymer composed of nucleotides.
  • a polynucleotide may be in the form of an individual fragment or may be a component of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration and is capable of passing standards Molecular biology methods (eg, using cloning vectors) identify, manipulate, and recover sequences and their component nucleotide sequences.
  • nucleotide sequence When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U” replaces "T".
  • polynucleotide refers to a polymer of nucleotides that has been removed from other nucleotides (individual fragments or entire fragments), or may be a component or component of a larger nucleotide structure, such as the expression Vector or polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences. "Recombinant polynucleotide” and “recombinant nucleic acid molecule” belong to one type of "polynucleotide”.
  • operably linked refers to a nucleic acid that is in a functional relationship with another nucleic acid.
  • first coding region and the second coding region are operably linked so that they are located in the same nucleotide chain.
  • Sequence identity and “percent identity” in this disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and comparing the aligned polynucleotides or polypeptides. The number of positions containing identical nucleotides or amino acid residues is scored and compared to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotides or amino acid residues.
  • Polynucleotides may differ at one position, for example, by containing different nucleotides (ie, substitutions or mutations) or missing nucleotides (ie, nucleotide insertions or nucleotide deletions in one or both polynucleotides).
  • Polypeptides may differ at one position, for example, by containing a different amino acid (ie, a substitution or mutation) or a missing amino acid (ie, an amino acid insertion or amino acid deletion in one or both polypeptides).
  • Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
  • two or more sequences or subsequences have at least 40%, 50%, 60% when compared and aligned with maximum correspondence using sequence comparison algorithms or measured by visual inspection. %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the "sequence" of nucleotide or amino acid residues Identity” or "Percent Identity”.
  • the determination/calculation of "sequence identity” or “percent identity” can be based on any suitable region of the sequence.
  • sequences are substantially identical throughout the entire length of either or both compared biopolymers (that is, nucleic acids or polypeptides).
  • coding region refers to a gene sequence capable of transcribing messenger RNA and ultimately translating it into a target polypeptide or protein.
  • upstream or downstream refer to both upstream and downstream in the direction of protein translation from a coding region.
  • the term "recombinant nucleic acid molecule” refers to a polynucleotide having sequences that are not linked together in nature.
  • the recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell.
  • the polynucleotide is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide,” "recombinant protein,” “fusion protein,” etc.
  • vector refers to a DNA construct containing DNA sequences operably linked to appropriate control sequences for expression of a gene of interest in a suitable host.
  • the term "recombinant expression vector” refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide.
  • Recombinant expression vectors may include, for example, i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) structural or coding sequences that are transcribed into mRNA and translated into proteins; and iii) appropriate transcription and transcriptional subunits of translation initiation and termination sequences.
  • Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons.
  • Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids and vectors derived from combinations of plasmid and phage DNA, from e.g. lentivirus, DNA from viruses such as retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.
  • viral plasmids such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids and vectors derived from combinations of plasmid and phage DNA, from e.g. lentivirus, DNA from viruses such as retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.
  • viral plasmids such as viral plasmids,
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
  • recombinant host cell covers a host cell that is different from the parent cell after the introduction of a recombinant nucleic acid molecule or a recombinant expression vector.
  • the recombinant host cell is specifically achieved by transformation.
  • the host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing the recombinant nucleic acid molecule or recombinant expression vector of the present disclosure.
  • a "host cell” in this disclosure is derived from a yeast cell.
  • Yeast saccharomyces cerevisiae.
  • the term "recombinant yeast strain” is obtained by modifying yeast cells using genetic engineering methods.
  • Embodiments include, but are not limited to, introducing recombinant genes, knocking out, and knockdown treatment of endogenous genes of yeast.
  • the term "recombinant gene” refers to a gene that does not occur naturally, and a recombinant gene includes a protein-coding sequence operably linked to an expression control sequence.
  • Embodiments include, but are not limited to, exogenous genes introduced into a microorganism, endogenous protein-coding sequences operably linked to heterologous promoters, and genes with modified protein-coding sequences.
  • Recombinant genes are stored in the genome of microorganisms, plasmids in microorganisms, or phages in microorganisms.
  • SPS Stretal Pressure Selection
  • SPS System The survival pressure selection system in the present disclosure involves a composition of fusion protein I and fusion protein II that converts the GPCR drug activation process into a cell growth process, a recombinant nucleic acid molecule encoding the composition of the fusion protein, a recombinant expression vector, and a recombinant yeast cell etc.
  • transformation, transfection, and transduction have meanings generally understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the methods of transformation, transfection, and transduction include any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microbial Injection method, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.
  • the methods disclosed herein may be performed in vitro, ex vivo, or in vivo, or the products may be present in in vitro, ex vivo, or in vivo forms.
  • in vitro refers to experiments using materials, biological substances, cells and/or tissues in laboratory conditions or culture media; whereas the term “in vivo” refers to experiments and procedures using intact multicellular organisms.
  • methods performed in vivo can be performed on non-human animals.
  • Ex vivo refers to an event that exists or occurs outside an organism, such as outside a human or animal body, such as an event that may exist or occur on tissue (eg, a whole organ) or cells taken from an organism.
  • the present disclosure provides a fusion protein composition, which includes the fusion protein I shown in (i) below and the following independently.
  • the first protein is selected from G protein-coupled receptors or functional variants thereof
  • the second protein is selected from G proteins or functional variants thereof
  • the first domain is selected from the carboxyl terminal structure of a reporter protein Domain
  • the second domain is selected from the amino-terminal domain of the reporter protein
  • the carboxyl-terminal domain of the reporter protein and the amino-terminal domain of the reporter protein are used to form a reporter protein, and the reporter protein releases a reporter signal for detection.
  • the reporter protein is split into two parts: a first domain (that is, the carboxyl-terminal domain of the reporter protein) and a second domain (that is, the amino-terminal domain of the reporter protein); and the GPCR or The functional variant thereof is fused to one of the first structural domain and the second structural domain, and the G protein or the functional variant thereof is fused to the second structural domain and the other one of the first structural domain and the second structural domain, Fusion protein I and fusion protein II were obtained.
  • the GPCR When the GPCR binds to the drug to be screened and is activated, it binds to the G protein, bringing the amino-terminal domain and carboxyl-terminal domain of the reporter protein close to each other, combining to form a functional reporter protein, which feeds back a detectable reporter signal.
  • the fusion protein I and fusion protein II designed based on protein fragment complementation in this disclosure innovatively split the reporter protein into two parts and fused them to the target GPCR and G protein, converting the GPCR activation signal into a report Reporter signal produced by the protein.
  • the reporter signal comes directly from the interaction between GPCR and G protein without going through a series of transcriptional regulation and cascade amplification, the signal is more authentic and credible, has the advantages of high sensitivity and reliability, and is suitable for GPCRs with low affinity or weak activation. GPCR drug screening.
  • the GPCR when a GPCR agonist is present in the environment, the GPCR can be activated to form a functionally active reporter protein. Then when there is a GPCR antagonist or GPCR reverse inhibitor in the environment, the GPCR antagonist or GPCR reverse inhibitor competitively binds to the GPCR with the GPCR agonist, causing the activated GPCR to be inhibited, the reporter protein loses its reporting activity, and feedback activation GPCR is inhibited information.
  • the first protein is selected from GPCRs or functional variants thereof. Since there are many types of GPCRs and they are widely distributed in various species, there is no restriction on the type of GPCR used to form fusion protein I, as long as it can be combined with the drug to be screened, so that the GPCR is activated and further binds to the G protein.
  • the screening drug signal can be converted into a reporting signal.
  • the GPCR of fusion protein I combines with the drug to be screened, changes from the activated state to the inhibitory state, releases the binding between the GPCR and the G protein, and feeds back the action information of the drug to be screened.
  • the GPCR is a human protein.
  • GPCR types include, but are not limited to, MC1R, MC4R, ⁇ 2 AR, etc.
  • the first protein used to form fusion protein I includes but is not limited to MC1R or its functional variants, MC4R or its functional variants, ⁇ 2 AR or its functional variants, etc.
  • the first protein is selected from ⁇ 2 AR or functional variants thereof.
  • ⁇ 2 AR is one of the currently widely used GPCRs.
  • Using ⁇ 2 AR or its functional variant as the first protein is suitable for extensive screening and identification of GPCR drugs.
  • the first protein is ⁇ 2AR .
  • the amino acid sequence of ⁇ 2 AR is as shown in SEQ ID NO:5, or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with ⁇ 2 AR Sequence of protein activity.
  • the first protein is a carboxy-terminally truncated [beta ]2AR .
  • the amino acid sequence of the carboxy-terminal truncated ⁇ 2 AR is as shown in SEQ ID NO: 6, or has an amino acid sequence of 80%, 81%, 82%, 83%, or 84% with the amino acid sequence shown in SEQ ID NO: 6 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and has a sequence with ⁇ 2 AR protein activity.
  • the first protein is MC4R.
  • Human melanocortical receptor-4 belongs to class A GPCR and is widely distributed in the central nervous system of the human body 15 , regulating human appetite and sexual behavior 16,17 . Studies have shown that activating MC4R can increase the body's feeling of fullness and help reduce food intake18 .
  • the drug is expensive, can only be administered by subcutaneous injection, and has strong side effects20 .
  • the amino acid sequence of MC4R is as shown in SEQ ID NO:7, or has 80%, 81%, 82%, 83%, 84%, 85%, 86% with the amino acid sequence shown in SEQ ID NO:7 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity and having MC4R protein activity .
  • the first protein is MC1R.
  • the amino acid sequence of MC1R is as shown in SEQ ID NO:8, or has 80%, 81%, 82%, 83%, 84%, 85%, 86% with the amino acid sequence shown in SEQ ID NO:8 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity and having MC1R protein activity .
  • the second protein is selected from G proteins or functional variants thereof.
  • the present disclosure does not limit the type and sequence of the second protein, as long as it can bind to the activated first protein in fusion protein I, so that the first domain and the second domain are brought close to each other and combined to become functionally active. Just report the protein.
  • the present disclosure selects protein-engineered small G protein (miniG) 13,14 as the second protein to simulate the biological function of the trimeric G protein for use with the reporter protein
  • miniG protein-engineered small G protein
  • the first domain or the second domain are fused to form fusion protein II.
  • the amino acid sequence of the miniG protein is as shown in SEQ ID NO:9, or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and have Sequence of miniG protein activity.
  • the reporter protein is a protein associated with survival.
  • the reporter protein is a key enzyme in a metabolic pathway encoded by an auxotrophic selection marker gene.
  • the auxotrophic screening marker gene is an important genetic screening marker.
  • the auxotrophic screening marker gene is related to the expression of key enzymes in the metabolic pathway and affects the growth and metabolic rate of microorganisms. Due to the wide variety of auxotrophic screening markers, the present disclosure does not limit the type of reporter protein encoded by the auxotrophic screening marker gene, as long as the reporter protein can compensate for the auxotrophy of the organism or cell and maintain the organism or cell. Normal growth in auxotrophic media is sufficient.
  • the key enzymes in the metabolic pathway encoded by the auxotrophic selection marker gene are ⁇ -isopropylmalate dehydrogenase, orotate-5'-phosphate decarboxylase, and phosphoribosyl aminoimidazole carboxylase , imidazole glycerol phosphate dehydratase, phosphoribosyl anthranilate isomerase, etc.
  • the reporter protein is ⁇ -isopropylmalate dehydrogenase (3-isopropylmalate dehydrogenase).
  • ⁇ -isopropylmalate dehydrogenase is encoded by the LEU2 gene and is a leucine synthesis pathway. key enzymes in. In yeast, the absence of ⁇ -isopropylmalate dehydrogenase results in the inability to synthesize leucine, making yeast unable to grow in media lacking leucine.
  • the reporter protein is Orotidine-5'-phosphate (OMP) decarboxylase, which is encoded by the URA3 gene and is a key enzyme in the uracil synthesis pathway. .
  • OMP Orotidine-5'-phosphate
  • the reporter protein is phosphoribosylaminoimidazole carboxylase, which is encoded by the Ade2 gene and is a key enzyme in the adenine synthesis pathway.
  • yeast the absence of phosphoribosaminoimidazole carboxylase results in the inability to synthesize adenine, making yeast unable to grow in adenine-deficient media.
  • the reporter protein is imidazoleglycerol-phosphate dehydratase, which is encoded by the His3 gene and is a key enzyme in the histidine synthesis pathway.
  • the absence of imidazole glycerol phosphate dehydratase results in the inability to synthesize histidine, making yeast unable to grow in media lacking histidine.
  • the present disclosure selects key enzymes in the tryptophan synthesis pathway as the reporter protein. Specifically, the present disclosure selects phosphoribosyl anthranilate isomerase (Trp1p) as the reporter protein, and the Trp1p protein is encoded by the gene encoding phosphoribosyl anthranilate isomerase (trp1).
  • Trp1p phosphoribosyl anthranilate isomerase
  • trp1 is a common auxotrophic screening marker gene in yeast biology. This gene encodes a phosphoribosyl anthranilate isomerase and is involved in the third step of tryptophan biosynthesis in Saccharomyces cerevisiae 11 . Yeast deficient in the trp1 gene cannot survive in tryptophan-deficient media due to the lack of the ability to synthesize tryptophan.
  • PDB id: 1LBM phosphoribose anthranilate isomerase
  • Trp1p is divided into two protein domains: a first domain and a second domain.
  • the first domain is the carboxy-terminal domain (Trp1p CTD )
  • the second domain is the amino-terminal domain (Trp1p NTD ).
  • One of the first domain and the second domain is fused with the first protein to form fusion protein I
  • the other of the first domain and the second domain is fused with the second protein to form fusion protein II.
  • the two protein domains in Fusion Protein I and Fusion Protein II will not spontaneously come together and assemble into functional proteins. They will only be recruited to the nearby GPCR receptor when a specific GPCR receptor is activated and binds to the G protein. The location thus restores its biological function, and this difference in function can significantly affect the growth rate of the organism (e.g., yeast).
  • first structural domain and the second structural domain formed by dividing Trp1p there is no restriction on the first structural domain and the second structural domain formed by dividing Trp1p, as long as the first structural domain and the second structural domain can form functionally active Trp1p when close to each other.
  • the amino acid sequence of the first domain is as shown in SEQ ID NO:3, or is 80%, 81%, 82%, 83%, 84% identical to the amino acid sequence shown in SEQ ID NO:3 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:3.
  • the amino acid sequence of the second domain is as shown in SEQ ID NO: 1, or is 80%, 81%, 82%, 83%, 84% identical to the amino acid sequence shown in SEQ ID NO: 1 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:1.
  • the reporter protein is a resistance protein.
  • Resistance proteins enable organisms to grow in media containing inhibitory drugs by hydrolyzing or assisting organisms (e.g., microorganisms such as yeast) to resist drugs that inhibit growth.
  • the reporter protein is aminoglycoside 3'-phosphotransferase, which is encoded by the Tn903 gene and can phosphorylate the antibiotic G418 to allow microorganisms such as yeast to grow in the antibiotic added with G418.
  • the reporter protein is engineered dihydrofolate reductase, encoded by the engineered DHFR gene.
  • the wild-type dihydrofolate reductase in yeast will be inhibited by methotrexate (also known as methotrexate), which leads to the nucleotide synthesis and growth of yeast.
  • methotrexate also known as methotrexate
  • the engineered dihydrofolate reductase can tolerate the effects of methotrexate. The inhibitory effect can thus help yeast growth [25] .
  • Fusion Protein I is formed from a first protein fused to a first domain.
  • the first protein is selected from GPCR or its functional variant, and the first domain is selected from the carboxyl terminal domain of the reporter protein.
  • Fusion Protein I is formed from a first protein fused to a second domain.
  • the first protein is selected from GPCR or its functional variant, and the second domain is selected from the amino-terminal domain of the reporter protein.
  • the first domain or the second domain can be fused to any fusion-able position within the first protein or at the carboxyl terminus. Specifically, it is sufficient to avoid fusion with the amino terminus of the first protein and allow the amino terminus of the first protein to bind to the GPCR drug to be screened outside the cell membrane.
  • fusion protein I is formed by fusion of the first domain to the carboxyl terminus of the first protein; exemplarily, as shown in c in Figure 1, the carboxyl terminus domain of the reporter protein is fused to GPCR or The carboxyl terminus of its functional variant forms fusion protein I.
  • the fusion protein I is formed by fusing the first structural domain to the interior of the first protein; for example, as shown in Figure 8, there is a flexible ICL3 region inside the GPCR or its functional variant. , the carboxyl-terminal domain of the reporter protein is fused to the ICL3 region to form fusion protein I.
  • the fusion protein II is formed by fusing the second structural domain to the carboxyl terminus of the first protein; in some other optional embodiments, the fusion protein II is composed of the second structural domain fused to the first protein. Internal formation of protein.
  • Fusion Protein I also includes a linking peptide for connecting the first protein to the first domain or the second domain.
  • the linker peptide is a GS linker peptide consisting of a stretch of glycine and serine residues.
  • the disclosure uses ⁇ 2AR as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • Trp1p CTD carboxyl-terminal domain of Trp1p
  • the present disclosure fuses Trp1p CTD to the carboxyl terminus of ⁇ 2 AR to form fusion protein I.
  • the structure of fusion protein I is (j 1 ) (N)- ⁇ 2 AR-Trp1p CTD -(C).
  • fusion protein I also includes a linker peptide connecting ⁇ 2 AR and Trp1p CTD .
  • the structure of fusion protein I is (j 3 ) (N)- ⁇ 2 AR-linked peptide-Trp1p CTD- (C).
  • the disclosure uses ⁇ 2AR as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • Trp1p CTD is fused to the interior of ⁇ 2 AR to form fusion protein I.
  • ⁇ 2 AR is divided into ⁇ 2 AR protein fragment I and ⁇ 2 AR protein fragment II along the direction from the amino terminus (N) to the carboxyl terminus (C).
  • the structure of fusion protein I is (j 5 ) (N)- ⁇ 2 AR protein fragment I-Trp1p CTD - ⁇ 2 AR protein fragment II-( C).
  • fusion protein I also includes connecting peptides connecting Trp1p CTD to ⁇ 2 AR protein fragment I and ⁇ 2 AR protein fragment II respectively.
  • the structure of fusion protein I is (j 7 ) (N)- ⁇ 2 AR protein fragment I-linking peptide-Trp1p CTD -linking peptide- ⁇ 2 AR protein fragment II-(C).
  • the present disclosure further truncates the carboxy terminus of ⁇ 2 AR, and uses the carboxy terminus truncated ⁇ 2 AR as the first protein ( For example, the region after the palmitoylation site C341 8.59 of the ⁇ 2 AR carboxyl terminus is truncated ( ⁇ 2 AR truncated fragment) to limit the position of the Trp1p CTD near the receptor.
  • Trp1p CTD carboxyl-terminal domain of Trp1p
  • the structure of fusion protein I is (j 2 )(N)-carboxyl terminus truncated ⁇ 2 AR-Trp1p CTD -(C).
  • Fusion Protein I also includes a linker peptide connecting the carboxyl-terminally truncated ⁇ 2 AR to the Trp1p CTD .
  • the structure of fusion protein I is (j 4 ) (N)-carboxyl terminus truncated ⁇ 2 AR-linked peptide-Trp1p CTD -(C) .
  • the amino acid sequence of fusion protein I is as shown in SEQ ID NO:10, or has 80%, 81%, 82%, 83%, 84% similarity with the amino acid sequence shown in SEQ ID NO:10 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:10.
  • the nucleotide sequence encoding fusion protein I is as shown in SEQ ID NO:18, or has 80%, 81%, or 82% similarity with the nucleotide sequence as shown in SEQ ID NO:18 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence having a protein activity as shown in SEQ ID NO: 10.
  • the present disclosure uses a carboxy-terminally truncated ⁇ 2 AR as the first protein and the carboxy-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • the Trp1p CTD was fused to the interior of the carboxyl-terminal truncated ⁇ 2 AR to form fusion protein I.
  • the carboxyl-terminal truncated ⁇ 2 AR is divided into carboxyl-terminal truncated ⁇ 2 AR along the direction from the amino terminus (N) to the carboxyl terminus (C). Protein fragment I and carboxyl-terminally truncated ⁇ 2 AR protein fragment II.
  • the structure of the fusion protein I is (j 6 ) (N)-carboxyl terminus truncated ⁇ 2 AR protein fragment I-Trp1p CTD –carboxyl terminus truncation Short ⁇ 2AR protein fragment II-(C).
  • fusion protein I also includes connecting peptides connecting Trp1p CTD to carboxyl-terminal truncated ⁇ 2 AR protein fragment I and carboxyl-terminal truncated ⁇ 2 AR protein fragment II respectively.
  • the structure of fusion protein I is (j 8 ) (N)-carboxyl terminus truncated ⁇ 2 AR protein fragment I-linking peptide-Trp1p CTD - Linking peptide-carboxy terminally truncated ⁇ 2AR protein fragment II-(C).
  • the present disclosure selects MC4R as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • Trp1p CTD carboxyl-terminal domain of Trp1p
  • the structure of fusion protein I is (j 9 ) (N)-MC4R-Trp1p CTD - (C).
  • fusion protein I also includes a connecting peptide connecting MC4R and Trp1p CTD .
  • the structure of fusion protein I is (j 10 ) (N)-MC4R-linking peptide-Trp1p CTD- (C).
  • the amino acid sequence of fusion protein I is as shown in SEQ ID NO:11, or has an amino acid sequence of 80%, 81%, 82%, 83%, 84% with the amino acid sequence shown in SEQ ID NO:11. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:11.
  • the nucleotide sequence encoding fusion protein I is as shown in SEQ ID NO:19, or has 80%, 81%, or 82% of the nucleotide sequence as shown in SEQ ID NO:19 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence having a protein activity as shown in SEQ ID NO: 11.
  • the present disclosure uses MC4R as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • Trp1p CTD is fused to the interior of MC4R to form fusion protein I.
  • MC4R is divided into MC4R protein fragment I and MC4R protein fragment II along the amino terminus (N) to the carboxyl terminus (C).
  • fusion protein I is (j 11 ) (N)-MC4R protein fragment I-Trp1p CTD -MC4R protein fragment II-(C).
  • fusion protein I also includes connecting peptides connecting Trp1p CTD to MC4R protein fragment I and MC4R protein fragment II respectively.
  • fusion protein I is (j 12 ) (N)-MC4R protein fragment I-linking peptide-Trp1p CTD -linking peptide-MC4R protein fragment II -(C).
  • Trp1p CTD is fused to the flexible ICL3 region on MC4R to form fusion protein I.
  • the amino acid sequence of fusion protein I is as shown in SEQ ID NO:13, or has an amino acid sequence of 80%, 81%, 82%, 83%, 84% with the amino acid sequence shown in SEQ ID NO:13. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:13.
  • the nucleotide sequence encoding fusion protein I is as shown in SEQ ID NO:21, or has 80%, 81%, 82%, or 83% similarity with the amino acid sequence shown in SEQ ID NO:21 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence Identity, and a nucleotide sequence encoding a protein with protein activity as shown in SEQ ID NO:13.
  • the present disclosure selects MC1R as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • Trp1p CTD carboxyl-terminal domain of Trp1p
  • the structure of fusion protein I is (j 13 ) (N)-MC1R-Trp1p CTD -(C).
  • fusion protein I also includes a connecting peptide connecting MC1R and Trp1p CTD .
  • the structure of fusion protein I is (j 14 ) (N)-MC1R-linking peptide-Trp1p CTD- (C).
  • the amino acid sequence of fusion protein I is as shown in SEQ ID NO:12, or has an amino acid sequence of 80%, 81%, 82%, 83%, 84% with the amino acid sequence shown in SEQ ID NO:12. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:12.
  • the nucleotide sequence encoding fusion protein I is as shown in SEQ ID NO:20, or has 80%, 81%, or 82% of the nucleotide sequence as shown in SEQ ID NO:20 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence having a protein activity as shown in SEQ ID NO: 12.
  • the present disclosure uses MC1R as the first protein and the carboxyl-terminal domain of Trp1p (Trp1p CTD ) as the first domain.
  • the Trp1p CTD is fused to the interior of MC1R to form fusion protein I.
  • MC1R is divided into MC1R protein fragment I and MC1R protein fragment II along the amino terminus (N) to the carboxyl terminus (C).
  • fusion protein I is (j 15 ) (N)-MC1R protein fragment I-Trp1p CTD -MC1R protein fragment II-(C).
  • fusion protein I also includes connecting peptides connecting Trp1p CTD to MC1R protein fragment I and MC1R protein fragment II respectively.
  • the structure of fusion protein I is (j 16 ) (N) - MC1R protein fragment I - connecting peptide - Trp1p CTD - connecting peptide - MC1R protein fragment II -(C).
  • fusion protein II is formed from a second protein fused to a second domain.
  • the second protein is selected from G proteins or functional variants thereof, and the second domain is selected from the amino-terminal domain forming the reporter protein.
  • fusion protein II is formed from a second protein fused to a second domain.
  • the second protein is selected from G proteins or functional variants thereof, and the second domain is selected from the amino-terminal domain forming the reporter protein.
  • the first domain or the second domain can be fused to any fusion-able position within the second protein or at the amino terminus. Since the carboxyl terminus of the second protein is critical for its interaction with the receptor, it needs to be preserved in the structure. Therefore, it is sufficient to avoid fusing the first domain or the second domain to the carboxyl terminus of the second protein so that the second protein can bind to the activated first protein.
  • fusion protein II is formed by fusing the second structural domain to the amino terminus of the second protein; in other optional embodiments, fusion protein II is formed by fusing the second structural domain to the second protein. In some other optional embodiments, the fusion protein II is formed by fusing the first structural domain to the amino terminus of the second protein; in some other optional embodiments, the fusion protein II is composed of the first structural domain fused to the interior of the second protein.
  • fusion protein II also includes a linker peptide for connecting the second protein to the first domain or the second domain.
  • the linker peptide is a GS linker peptide consisting of a stretch of glycine and serine residues.
  • the present disclosure uses miniG as the second protein and the amino-terminal domain of Trp1p (Trp1p NTD ) as the first domain. Trp1p NTD was fused to the amino terminus of miniG to form fusion protein II.
  • the structure of fusion protein II is (k 3 ) (N)-Trp1p NTD -miniG protein-(C).
  • the fusion protein II also includes a connecting peptide connecting miniG and Trp1p NTD .
  • the structure of fusion protein I is (k 4 ) (N)-Trp1p NTD -linking peptide-miniG protein-(C).
  • the present disclosure uses miniG as the second protein and the amino-terminal domain of Trp1p (Trp1p NTD ) as the first domain.
  • Trp1p NTD is fused to the interior of miniG to form fusion protein II.
  • miniG is divided into miniG protein fragment I and miniG protein fragment II along the direction from the amino terminus (N) to the carboxyl terminus (C).
  • the structure of fusion protein II is (k 1 )(N)-miniG protein fragment I-Trp1p NTD -miniG protein fragment II-(C).
  • fusion protein II also includes connecting peptides connecting Trp1p NTD to miniG protein fragment I and miniG protein fragment II respectively.
  • the structure of fusion protein II is (k 2 ) (N)-miniG protein fragment I-linking peptide-Trp1p NTD -linking peptide-miniG protein fragment II -(C).
  • the miniG protein model comes from the protein structure with PDB ID 6GDG.
  • the schematic diagram of the miniG protein sequence and secondary structure is shown in Figure 6, which includes an ⁇ -helical structure, a ⁇ -sheet structure, and the rest is Flexible area.
  • Figure 6 shows multiple different fusion positions and nearby amino acid sequences in the miniG protein.
  • Three of the fusion sites are flexible regions with no specific secondary structure on the miniG protein structure, including the h4s6 region (site 1), the h3s5 region (site 2), and the flexible region before the N-terminus (site 3). and hgh4 flexible region (site 4).
  • the first domain or the second domain can be fused to miniG at any position from positions 1 to 4 to form fusion protein II.
  • Trp1p NTD is fused in a specific flexible region of the miniG protein, h4s6loop (site 1), to form fusion protein II.
  • Trp1p NTD is fused to a specific flexible region h4s6loop (site 1) of the miniG protein through a linker peptide to form fusion protein II.
  • the amino acid sequence of fusion protein II is as shown in SEQ ID NO:14, or has an amino acid sequence of 80%, 81%, 82%, 83%, 84% with the amino acid sequence shown in SEQ ID NO:14. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:14.
  • the nucleotide sequence encoding fusion protein II is as shown in SEQ ID NO:22, or has 80%, 81%, or 82% similarity with the nucleotide sequence as shown in SEQ ID NO:22 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence having a protein activity as shown in SEQ ID NO: 14.
  • Trp1p NTD is fused at site 2 in the miniG protein to form fusion protein II.
  • the Trp1p NTD is fused to site 2 of the miniG protein via a linker peptide to form fusion protein II.
  • the amino acid sequence of fusion protein II is as shown in SEQ ID NO:15, or has an amino acid sequence of 80%, 81%, 82%, 83%, 84% with the amino acid sequence shown in SEQ ID NO:15. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:15.
  • the nucleotide sequence encoding fusion protein II is as shown in SEQ ID NO:23, or has 80%, 81%, or 82% similarity with the nucleotide sequence as shown in SEQ ID NO:23 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence having a protein activity as shown in SEQ ID NO: 15.
  • Trp1p NTD is fused at site 3 in the miniG protein to form fusion protein II.
  • the Trp1p NTD is fused to site 3 of the miniG protein via a linker peptide to form fusion protein II.
  • the amino acid sequence of fusion protein II is as shown in SEQ ID NO:16, or has 80%, 81%, 82%, 83%, 84% similarity with the amino acid sequence shown in SEQ ID NO:16 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:16.
  • the nucleotide sequence encoding fusion protein II is as shown in SEQ ID NO:24, or has 80%, 81%, or 82% of the nucleotide sequence as shown in SEQ ID NO:24 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity, and encodes a nucleotide sequence with protein activity as shown in SEQ ID NO:16.
  • Trp1p NTD is fused at site 4 in the miniG protein to form fusion protein II.
  • the Trp1p NTD is fused to site 4 of the miniG protein via a linker peptide to form fusion protein II.
  • the amino acid sequence of fusion protein II is as shown in SEQ ID NO:17, or has 80%, 81%, 82%, 83%, 84% similarity with the amino acid sequence shown in SEQ ID NO:17 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, And has a sequence with protein activity as shown in SEQ ID NO:17.
  • the nucleotide sequence encoding fusion protein II is such as SEQ ID NO:25, or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity , and encodes a nucleotide sequence of a protein with protein activity as shown in SEQ ID NO:17.
  • Figure 8 shows another set of possible fusion positions of Trp1p CTD and Trp1 NTD on GPCRs and G proteins.
  • the flexible ICL3 region on GPCR is close to the hgh4 flexible region of G protein.
  • Trp1p CTD can be fused to the ICL3 region of the receptor
  • Trp1p NTD can be fused to the hgh4 region of G protein.
  • the present disclosure further provides a Survival Pressure Selection (SPS) system constructed from fusion protein I and fusion protein II.
  • SPS Survival Pressure Selection
  • fusion protein I and fusion protein II can be independently selected from any type of fusion protein mentioned above.
  • a in Figure 1 shows a schematic diagram of the model of the survival pressure screening system.
  • the GPCR receptor and miniG protein are respectively fused to the carboxyl-terminal domain (Trp1p CTD ) and the amino-terminal domain (Trp1p NTD ) of Trp1p and are expressed in the cell membrane and cytoplasm of Saccharomyces cerevisiae.
  • GPCR After GPCR is activated by an agonist, it binds to the intracellular miniG protein to promote the formation of functional Trp1p protein, allowing auxotrophic cells (for example, yeast cells) to restore their own ability to synthesize tryptophan, thereby achieving tryptophan deficiency. grown in culture medium.
  • auxotrophic cells for example, yeast cells
  • a GPCR agonist is first used to activate the GPCR, and then under treatment with the drug to be screened, if the activation effect of the GPCR is competitively weakened or eliminated, the growth rate of the auxotrophic cells will also be reduced or terminated accordingly.
  • FIG. 1 shows a schematic diagram of the structural design of fusion protein I and fusion protein II in the SPS system.
  • the Trp1p protein is split at the appropriate position and fused to the miniG protein and GPCR respectively.
  • GPCR forms a complex with miniG protein
  • the two separated parts of Trp1p can combine at the appropriate location near the complex to form a functional protein.
  • the positions where the receptor fuses with the Trp1p CTD and the position where the miniG protein fuses with the Trp1p NTD are circled by the solid and dotted lines in the figure respectively.
  • the present disclosure constructs a SPS system ( ⁇ 2 AR-SPS) based on ⁇ 2 AR, miniG protein, and Trp1p protein.
  • the present disclosure constructs an SPS system (MC4R-SPS) based on MC4R, miniG protein, and Trp1p protein.
  • the present disclosure constructs an SPS system (MC1R-SPS) based on MC1R, miniG protein, and Trp1p protein.
  • the SPS system in the present disclosure can also be formed by proteins encoded by other types of GPCRs, G proteins or auxotrophic screening marker genes.
  • the SPS system provided by this disclosure can be applied to other types of GPCRs through simple adjustments, and has broad promotion and application value.
  • the present disclosure further provides recombinant nucleic acid molecules expressing fusion proteins, which include nucleotide sequences encoding fusion protein I and fusion protein II, which can be used to express fusion protein I and fusion protein II in cells to construct fusion proteins for GPCR drug screening.
  • fusion proteins which include nucleotide sequences encoding fusion protein I and fusion protein II, which can be used to express fusion protein I and fusion protein II in cells to construct fusion proteins for GPCR drug screening.
  • Cell model recombinant nucleic acid molecules expressing fusion proteins, which include nucleotide sequences encoding fusion protein I and fusion protein II, which can be used to express fusion protein I and fusion protein II in cells to construct fusion proteins for GPCR drug screening.
  • a recombinant nucleic acid molecule includes operably linked elements shown below:
  • a first coding region comprising a first coding sequence encoding fusion protein I
  • a second coding region comprising a second coding sequence encoding fusion protein II.
  • the first coding sequence is a nucleotide sequence shown in any one of SEQ ID NO: 18-21, or a nucleoside sequence shown in any one of SEQ ID NO: 18-21 Acid sequence comparison has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% sequence identity, and encoding a nucleotide sequence having a protein activity encoded by the sequence shown in any one of SEQ ID NO: 18-21.
  • the second coding sequence is a nucleotide sequence shown in any one of SEQ ID NO:22-25, or a nucleoside sequence shown in any one of SEQ ID NO:22-25.
  • Acid sequence comparison has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% sequence identity, and encoding a nucleotide sequence having a protein activity encoded by the sequence shown in any one of SEQ ID NO: 22-25.
  • the recombinant nucleic acid molecule further includes one or more promoters operably linked to the first coding region or the second coding region.
  • the promoter is a bidirectional promoter connecting the first coding region and the second coding region.
  • the first coding region is located upstream of the bidirectional promoter and the second coding region is located downstream of the bidirectional promoter.
  • the second coding region is located upstream of the bidirectional promoter, and the first coding region is located downstream of the bidirectional promoter.
  • the bidirectional promoter can be of any type, and this disclosure is not restrictive.
  • a bidirectional promoter is GAL1,10.
  • the first coding region and the second coding region exist independently of each other.
  • the first coding region and the second coding region are located in two recombinant vectors respectively.
  • the recombinant yeast cells provided by the present disclosure express any one of the fusion protein compositions provided by the present disclosure, or the recombinant nucleic acid molecules containing any one of the fusion protein-encoding compositions provided by the present disclosure.
  • a type of reporter protein related to yeast survival is used in the present disclosure, and the reporter protein is encoded by an auxotrophic screening marker gene.
  • the GPCR activation process is transformed into A method of assisting yeast growth.
  • candidate compounds that activate specific GPCRs can lead to faster growth in yeast.
  • an activated GPCR when combined with a GPCR antagonist or GPCR reverse inhibitor, it can also competitively inhibit or eliminate the activation effect of GPCR, causing the yeast to slow down or terminate its growth rate.
  • this disclosure a variety of different survival-related proteins are experimentally verified as reporter proteins, including some antibiotic resistance proteins or key enzymes in metabolic pathways. After a series of screening and verification, this disclosure selected the key enzyme in the tryptophan synthesis pathway as the reporter protein. Furthermore, this disclosure selects phosphoribosyl anthranilate isomerase (Trp1p) as the reporter protein.
  • Trp1p phosphoribosyl anthranilate isomerase
  • Trp1p is divided into two protein domains: a first domain (Trp1p CTD ) and a second domain (Trp1p NTD ).
  • the two protein domains are fused with GPCR and G protein respectively to form fusion protein I and fusion protein II.
  • the two protein domains in Fusion Protein I and Fusion Protein II will not spontaneously come together and assemble into functional proteins. They will only be recruited to the nearby GPCR receptor when a specific GPCR receptor is activated and binds to the G protein. position thereby restoring its biological function, and this functional difference can significantly affect the growth rate of yeast.
  • fusion protein I and fusion protein II can be selected from any type of protein fusion protein I and fusion protein II described in this disclosure.
  • the present disclosure further constructs the recombinant yeast strain EBY100- ⁇ 2 AR-SPS based on the ⁇ 2 AR-SPS system.
  • the present disclosure further constructs the recombinant yeast strain EBY100-MC4R-SPS based on the MC4R-SPS system.
  • the present disclosure further constructs the recombinant yeast strain EBY100-MC1R-SPS based on the MC1R-SPS system.
  • This disclosure does not limit the type of GPCR. Specifically, as long as the interaction information between GPCR and the drug to be screened can be converted into a reporting signal released by the reporter protein through the combination of GPCR and G protein, by detecting the activity of recombinant yeast cells. Growth, it is enough to achieve highly sensitive and specific screening of the drugs to be screened.
  • the recombinant yeast cells of the present disclosure are derived from Saccharomyces cerevisiae cells.
  • Saccharomyces cerevisiae is a single-cell model organism that has been widely studied at present. Compared with mammalian cells or insect cells, using Saccharomyces cerevisiae as a host cell to construct a drug screening system has the following advantages: the yeast endogenous GPCR/G protein signaling pathway is almost completely orthogonal to the human GPCR/G protein signaling pathway, and can be used in False positive results caused by signal cross-linking responses are largely avoided6 . Secondly, GPCRs can be expressed in yeast and maintain their original functions7 . In addition, compared with mammalian culture, yeast culture is simple and fast, and yeast is more tolerant to commonly used organic solvents such as DMSO and ethanol.
  • Saccharomyces cerevisiae lacks human GPCR signal transduction proteins
  • the present disclosure is designed to use the specific interaction between the receptor and its signal transduction protein (such as G protein or arrestin protein) to develop a protein-fragment complementation method (protein-fragment complementation). assay, PCA) functional screening system.
  • the reporter protein will be divided into two parts at the appropriate position and fused and expressed on the two target proteins (prey protein and bait protein) respectively. The interaction between these two proteins will drive the two parts of the reporter protein closer to each other to form a The protein is functional and gives a detectable feedback signal.
  • the Saccharomyces cerevisiae cells provided by the present disclosure are auxotrophic Saccharomyces cerevisiae cells, and the reporter proteins used to form fusion protein I and fusion protein II in the recombinant yeast cells are encoded by auxotrophic screening marker genes. protein.
  • Recombinant yeast cells derived from auxotrophic Saccharomyces cerevisiae cells cannot grow normally in media lacking specific nutrients.
  • the GPCR in fusion protein I is combined with the drug to be screened and is activated, it can promote the combination in the recombinant yeast cells to form a functional
  • the active reporter protein makes up for the nutritional deficiencies of the recombinant yeast cells and significantly promotes the growth of the recombinant yeast cells.
  • the screening and verification of GPCR drugs can be achieved.
  • the activated GPCR is combined with the drug to be screened to inhibit the GPCR, which can competitively inhibit or eliminate the activation effect of the GPCR.
  • the activity of the reporter protein formed by the combination of fusion protein I and fusion protein II is lost, thereby reducing or terminating the recombinant yeast. Cell growth.
  • the S. cerevisiae cells provided by the present disclosure are tryptophan auxotrophic S. cerevisiae cells. In some more preferred embodiments, the S. cerevisiae cells provided by the present disclosure are trp1 gene-deficient S. cerevisiae cells.
  • the Saccharomyces cerevisiae cells provided by the present disclosure are non-auxotrophic Saccharomyces cerevisiae cells
  • the reporter proteins used to form fusion protein I and fusion protein II in the recombinant yeast cells are resistance proteins, and the resistance proteins can be decomposed Drugs that inhibit the growth of Saccharomyces cerevisiae cells, or drugs that assist Saccharomyces cerevisiae cells in resisting growth inhibition.
  • Recombinant yeast cells cannot grow normally in media containing specific drugs that inhibit their growth.
  • the GPCR in fusion protein I When the GPCR in fusion protein I is combined with the drug to be screened and is activated, it can promote the combination of the recombinant yeast cells to form a functionally active reporter protein, making the recombinant Yeast cells can resist growth inhibition and significantly promote the growth of recombinant yeast cells. By detecting the growth rate of recombinant yeast cells, the screening and verification of GPCR drugs can be achieved.
  • the recombinant yeast cells provided by the present disclosure establish a signal response system orthogonal to the human GPCR-G protein signaling pathway by expressing fusion protein I and fusion protein II in yeast cells.
  • Recombinant yeast cells can convert the activation process of GPCR into the growth process of yeast, producing a highly sensitive, direct and reliable growth reporting signal, which is suitable for the screening of drug molecules with low affinity to GPCR or weak activation effect, and is extremely useful in clinical drug screening. Has application prospects.
  • the GPCR-G protein signaling pathway constructed in yeast cells in the present disclosure does not interact with the yeast's own signal transduction pathway, and false positive results caused by signal cross-linking responses can be avoided to a large extent.
  • the final feedback signal of the survival reporter protein from the recombinant yeast cells only comes from the activation of the target GPCR and will not be interfered by other factors, avoiding the impact of the yeast's own stress response on the reporter system that may occur under growth pressure. , the system is more stable and reliable.
  • the recombinant yeast cells constructed in the present disclosure do not interfere with the genetic process of the yeast itself, and have no impact on the gene expression regulation required for its normal metabolism. Therefore, there is no need to carry out complex transformation and editing of the host yeast strain to avoid continuous activation of the yeast. A certain transcriptional regulation process has a serious impact on the normal life process of yeast, and the system is simpler.
  • the high-throughput screening device provided by the present disclosure includes:
  • Drug module used to store drugs to be screened
  • a strain inoculation module used to inoculate any one of the recombinant yeast cells of the present disclosure into the drug module, so that the recombinant yeast cells come into contact with the drug to be screened;
  • a culture module used for cultivating the recombinant yeast cells inoculated in the drug module
  • a measurement module used to obtain the growth rate of the recombinant yeast cells cultured in the culture module
  • a determination module is used to determine whether the drug to be screened is a target screening drug based on the growth rate of the recombinant yeast cells.
  • the high-throughput screening device can realize high-throughput screening of GPCR drugs, accurately identify agonists of target GPCRs in a larger screening range, and is useful for screening and discovering new agonists with lower affinity. With remarkable effect.
  • the high-throughput screening device further includes a transfer module for transferring the drug to be screened to the drug module.
  • the transfer module is the sonic liquid transfer system Echo550.
  • the drug module includes a multi-well plate with an array of microwells, the microwells are independently arranged on the multi-well plate, and the drugs to be screened are stored in a first preset number of the microwells. inside the hole.
  • the drug module may also include other types of storage mechanisms, specifically, as long as the storage of drugs to be screened can be achieved.
  • the first preset number in order to achieve high-throughput screening of drugs to be screened, can be set to at least 50, preferably at least 100, and more preferably at least 300.
  • the first preset number is 50, 55, 50, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 1000, etc.
  • the first preset number of microwells in the multi-well plate can be determined according to the number of types of drug molecules to be screened.
  • the microwells of the multi-well plate also contain a culture medium for cultivating recombinant yeast cells
  • the transfer module can be used to transfer the drug to be screened into the multi-well plate containing the culture medium in the microwells.
  • Echo550 can be used to transfer the drug to be screened stored in the first multi-well plate to a second multi-well plate containing culture medium in the microwells, where the drug to be screened is transferred to the second multi-well plate as the drug. module.
  • recombinant yeast cells are inoculated in a strain inoculation module into a drug module that transfers drug molecules to be screened, and the recombinant yeast cells are contacted with the drug molecules to be screened.
  • the SPS system in the recombinant yeast cells interacts with the drug molecules to be screened, if the GPCR in the SPS system is activated, it can effectively promote the growth of the recombinant yeast cells and convert the drug effect information of the drugs to be screened into the growth information of the recombinant yeast cells.
  • the measurement module is used to obtain the OD value of the recombinant yeast cells after culture in the culture module.
  • the measurement module is a microplate reader capable of detecting changes in OD values in microwells in a multi-well plate. By detecting the OD value in the microwell, the growth rate of the recombinant yeast cells can be fed back, and thus the GPCR activation effect of the drug to be screened can be fed back.
  • the determination module is used to determine whether the drug to be screened is a GPCR agonist based on the OD value of the recombinant yeast cell. For example, in the determination module, the OD 600 of the recombinant yeast cells after contact with the drug to be screened is calculated and counted, and the growth curves of the recombinant yeast cells in each well are plotted together, where the growth rate is expressed as the fold change of OD 600 . ( Figure 5b).
  • the present disclosure establishes a high-throughput screening platform for GPCR agonists.
  • the small molecule compounds stored in the 384-well plate were transferred to a sterile new 384-well plate with nanoscale precision using sonic transfer technology 23 , and the constructed EBY100-GPCR-SPS yeast was inoculated into the 384-well plate.
  • small molecule compounds that can increase the growth rate of yeast can be identified, and the ability of the screened compounds to activate specific GPCRs can be further verified through established biochemistry or cell biology experiments. Biological functions for further optimization.
  • the present disclosure further constructs a high-throughput screening system (which can also become a high-throughput screening platform) for specific GPCR targets, which is of great significance for the research and development of new drugs.
  • This disclosure used this system to screen approximately 10,000 compound molecules for human melanocortin 4receptor (MC4R), and successfully discovered new agonist molecules that can specifically activate MC4R at the cellular level. More importantly, this small molecule can only activate MC4R at micromolar concentrations, which directly proves the potential of the SPS system for finding weaker candidate compounds.
  • M4R human melanocortin 4receptor
  • the high-throughput drug screening device based on the SPS system in the present disclosure can find new agonists, antagonists, reverse inhibitors or forward allosteric modulating compounds of potential GPCR targets simply and at low cost. Since this method compares the difference in yeast growth rate within about a week under the treatment of different small molecule compounds, the results will have a strong signal-to-noise ratio and reliability, and can help researchers find some weaker candidate compounds. . These weaker candidate compounds often have novel backbones, binding sites, or binding modes compared to other known drugs for the target, and these new properties may demonstrate some biological properties of greater pharmacological value. Functions, such as better selectivity or preference for agonism, have the potential to be developed into new small molecule drugs with clinical value.
  • the drug screening methods provided by this disclosure include:
  • Cultivating step cultivating any one of the recombinant yeast cells of the present disclosure in a screening culture
  • Drug treatment step use the drug to be screened to treat the recombinant yeast cells
  • Comparing step Compare the growth rate of the recombinant yeast cells treated with the drug with the growth rate of the recombinant yeast cells not treated with the drug;
  • Determining step Based on the growth rate of the recombinant yeast cells, determine whether the drug to be screened is the target screening drug.
  • the screening medium is a medium used to screen for GPCR agonists.
  • the medium used for screening GPCR agonists is an auxotrophic medium
  • the recombinant yeast cells cultured in the screening medium are constructed based on a reporter protein encoded by an auxotrophic screening marker gene.
  • the determination step if the growth rate of the recombinant yeast cells after drug treatment is higher than the growth rate of the recombinant yeast cells without drug treatment, it is judged that the drug to be screened is a GPCR agonist.
  • the screening medium is a medium containing a drug for inhibiting yeast growth
  • the recombinant yeast cells cultured in the screening medium are based on resistance to the drug that inhibits yeast growth. Protein construction is formed.
  • the screening medium is a medium used for screening for GPCR antagonists or reverse inhibitors.
  • the medium used for screening GPCR antagonists or reverse inhibitors includes: auxotrophic medium and a GPCR agonist.
  • the determination step if the growth rate of the recombinant yeast cells after drug treatment is lower than the growth rate of the recombinant yeast cells without drug treatment, it is judged that the drug to be screened is a GPCR antagonist or reverse inhibitor.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • the Saccharomyces cerevisiae strain used in this disclosure is EBY100.
  • the commercial yeast plasmid pYD1 is used as the plasmid backbone and transformed into pYD-SPS-b2AR expressing fusion protein I and fusion protein II in EBY100.
  • fusion protein I is formed by fusion of Trp1p CTD to the carboxyl terminus of ⁇ 2 AR;
  • fusion protein II is formed by fusion of Trp1p NTD to the interior of miniGs protein.
  • the specific construction process is as follows:
  • the EBY100 strain has two genetic transformation selection markers, leucine (LEU2) and tryptophan (TRP1).
  • the pYD1 plasmid contains the TRP1 selectable tag for transformation screening of Saccharomyces cerevisiae. First, use the pYD1 plasmid as a template, use primer 1 and primers to perform PCR amplification, replace the TRP1 selective tag in the pYD1 plasmid with the LEU2 selective tag, and obtain the recombinant plasmid pYD-SPS.
  • the pYD-SPS plasmid contains an inducible bidirectional promoter GAL1,10, which can induce the expression of two different types of proteins on both sides in the presence of galactose (hereinafter distinguished by "downstream” and “upstream”
  • the promoter drives expression in both directions).
  • Use primers 3 and 4 to amplify gene fragment 1 of ⁇ 2 AR-first connecting peptide (the first connecting peptide serves as a flexible linking region and can be fused to the carboxyl terminus of ⁇ 2 AR protein), and use reverse amplification PCR to amplify the gene fragment 1 1 is inserted downstream of the promoter GAL1,10; use primer 3 and primer 4 to amplify gene fragment 2 expressing Trp1p CTD using primer 5 and primer 6 and insert it downstream of gene fragment 1 using reverse amplification PCR so that Trp1p CTD is fused to the carboxyl terminus of the ⁇ 2 AR-first connecting peptide; forming the first coding region downstream of the promoter, the first coding region is used to encode fusion protein I formed by the fusion of Trp1p CTD and ⁇ 2 AR.
  • This example constructs pYD-SPS-MC4R for expressing fusion protein I and fusion protein II; wherein, fusion protein I is formed by fusion of Trp1p CTD to the carboxyl terminus of MC4R; fusion protein II is formed by fusion of Trp1p NTD within the miniGs protein. .
  • Example 1 The plasmid construction method in Example 1 was adopted, and the gene fragment 1 expressing ⁇ 2 AR-first connecting peptide in the recombinant plasmid was replaced with the gene fragment 5 expressing MC4R; the gene fragment 2 was inserted downstream of the gene fragment 5 to form a promoter.
  • the first coding region downstream of the subunit is used to encode the fusion protein I formed by Trp1p CTD and MC4R.
  • Other parts of the recombinant plasmid remain the same as in Example 1.
  • the specific primers used during the construction process are as follows:
  • Example 3 Construction of recombinant plasmids pYD-SPS-MC1R-p3 and pYD-SPS-MC1R-p2
  • This example constructs pYD-SPS-MC1R for expressing fusion protein I and fusion protein II; wherein, fusion protein I is formed by fusion of Trp1p CTD to the carboxyl terminus of MC1R; fusion protein II is formed by fusion of Trp1p NTD within the miniGs protein. .
  • the plasmid construction method in Example 1 was adopted, and the gene fragment 1 expressing ⁇ 2 AR-first connecting peptide in the recombinant plasmid was replaced with the gene fragment 6 expressing MC1R; the gene fragment 2 was inserted downstream of the gene fragment 6 to form a promoter.
  • the first coding region downstream of the subunit is used to encode the fusion protein I formed by Trp1p CTD and MC1R; the specific primers used in the construction process are as follows:
  • primers 15 and 16 were used, and primers 17 and 18 were used to amplify Trp1p NTD gene fragments 7 (position 3, p3) and 8 (position 2, respectively).
  • p2 insert it into a specific position within gene fragment 3, so that Trp1p NTD is fused to a specific position of the miniGs protein to form a second coding region located upstream of the promoter.
  • the second coding region is used to encode the fusion formed by the fusion of Trp1pNTD and miniGs. Protein II.
  • the specific primers used during the construction process are as follows:
  • recombinant plasmid III pYD-SPS-MC1R-p3, position 3, EBY100-MC1R-SPS-p3
  • recombinant plasmid IV pYD-SPS-MC1R- p2, position 2, EBY100-MC1R-SPS-p2
  • This example was modified based on the existing pYD-SPS-MC4R plasmid, changing the fusion position of Trp1p CTD on MC4R and the fusion position of Trp1p NTD on miniG protein to construct the recombinant plasmid pYD-SPS-MC4R-p2.
  • primers were used to delete the Trp1p CTD originally fused to the carboxyl terminus of MC4R and the Trp1p NTD fused to the first site of the miniG protein on the plasmid.
  • primer 19 and primer 20 were used to reversely amplify plasmid pYD-SPS-MC4R, and the digested product was digested with DPN1 at 37 degrees Celsius for one hour and then transformed into E. coli top10 competent cells.
  • single clones were picked for sequencing, and the correctly sequenced plasmid was selected as the template for the next step.
  • the Trp1p CTD originally fused to the carboxyl terminus of MC4R had been deleted from the plasmid.
  • Use primer 21 and primer 22 to reversely amplify the new template plasmid, and use the same method to delete the Trp1p NTD originally fused at the first site of miniGs.
  • the specific primers used during the construction process are as follows:
  • Trp1p CTD was inserted into the specific region of the MC4R protein, and finally the constructed pYD-SPS-MC4R-p2 plasmid was obtained for growth testing.
  • the specific primers used during the construction process are as follows:
  • Saccharomyces cerevisiae is in a state where it is easy to accept external DNA into the cell.
  • This yeast competent state can be directly used for electroporation transformation or frozen through gradient cooling.
  • the recovered yeast was centrifuged at 3500g to remove the culture medium, and the cells were washed with leucine-deficient culture medium, spread on a leucine-deficient plate, and left to stand at 30°C for 48-72 hours to screen for transformants.
  • Example 6 Detection of the growth rate of recombinant yeast strain EBY100- ⁇ 2 AR-SPS
  • the recombinant plasmid pYD-SPS- ⁇ 2 AR prepared in Example 1 was transformed into yeast competent cells to obtain the EBY100- ⁇ 2 AR-SPS strain.
  • the constructed EBY100- ⁇ 2 AR-SPS strain was grown to saturated concentration in leucine-deficient culture medium and its OD 600 was measured.
  • the EBY100- ⁇ 2 AR-SPS strain showed significantly different growth rates under treatment with different ⁇ 2 AR agonists or antagonists (Fig. 2).
  • ⁇ 2 AR the Trp1p NTD is fused to a specific flexible region h4s6loop of the miniG protein through the connection of three amino acids of GGS, and the Trp1p CTD is fused to the carboxyl end of the receptor through the connection of 10 GS amino acids.
  • supplementing the ⁇ 2 AR full agonist BI-167107 in the tryptophan-deficient culture medium can help yeast grow at a significantly higher rate than the control group.
  • ⁇ 2 AR-SPS The combination of fusion protein I and fusion protein II constructed based on the ⁇ 2 AR target site was named ⁇ 2 AR-SPS, and the recombinant yeast strain expressing ⁇ 2 AR-SPS was named EBY100- ⁇ 2 AR-SPS.
  • this example uses the ⁇ 2 AR agonist isoprenaline (Isoprenaline) with a fast dissociation speed. and Epinephrine to test whether the growth rate of the EBY100- ⁇ 2 AR-SPS strain is agonist dependent.
  • the EBY100- ⁇ 2 AR-SPS yeast strain was cultured and its growth rate tested in the presence of different types and concentrations of agonists.
  • Yeast i.e., Apo
  • Ascorbic acid asorbic acid
  • yeast with only ascorbic acid added was set up as a control.
  • yeast Different groups of yeast were placed in 50 ml sterile centrifuge tubes, cultured with shaking at 25°C and 250 rpm, and their growth rates were measured to determine whether the rapid growth of EBY100- ⁇ 2 AR-SPS yeast depends on exogenous agonists.
  • Figure 3 shows the results of the agonist-dependent assay of EBY100- ⁇ 2 AR-SPS.
  • the grown yeast will be diluted into tryptophan-deficient culture medium containing agonists, antagonists or without any exogenous ligands and continued to be cultured.
  • the results showed that the EBY100- ⁇ 2 AR-SPS yeast that survived the first round of agonist stimulation could only continue to grow in the tryptophan-deficient culture medium supplemented with agonists, but could not grow in the tryptophan-deficient culture medium containing antagonists or without agonists. Produced in the culture medium of the body.
  • Example 8 Detection of the growth rate of recombinant yeast strain EBY100-MC4R-SPS
  • the recombinant plasmid pYD-SPS-MC4R prepared in Example 2 was transformed into yeast competent cells to obtain the EBY100-MC4R-SPS strain.
  • the constructed EBY100-MC4R-SPS strain was grown to saturated concentration in leucine-deficient culture medium and its OD 600 was measured.
  • Setmelanotide is dissolved in DMSO.
  • Different groups of yeast were cultured at 25 degrees Celsius and 250 rpm with shaking, and a spectrophotometer was used to measure the differences in the growth rates of the yeast in different groups.
  • EBY100-MC4R-SPS yeast can grow significantly faster than the control group under the action of the exogenous agonist Setmelanotide (SET) in tryptophan-deficient culture medium, and 10 ⁇ M Setmelanotide can increase the concentration of EBY100-MC4R-SPS in tryptophan 37 times that of the control group. Growth rate in acid-deficient culture medium, the growth rate is related to the concentration of agonist used ( Figure 4).
  • Setmelanotide Setmelanotide
  • the present disclosure successfully enables the rapid transfer of this method from ⁇ 2 AR to other GPCRs.
  • the published complex structures of GPCR and G protein21,22 prove that the interaction mode between GPCR and G protein is relatively conserved in different receptors. Therefore, the SPS system in the present disclosure is widely suitable for screening novel small molecule agonist drugs for GPCRs.
  • Small molecules were transferred to a sterile transparent 384-well plate using an Echo 550 nanoliter sonic pipetting system, with 50 nl of liquid transferred per well.
  • 384 plate 40 ⁇ l yeast culture medium per well. The plate is cultured in an incubator at a constant temperature and humidity of 25 degrees Celsius. Its OD 600 value is measured every day and the data is processed using a MATLAB script. The final data is presented by the fold change of OD 600. Based on the continuous culture results, the better small molecule compounds are selected. Biological activity functional verification is performed individually.
  • HEK293T cells were stably cultured at 37 degrees Celsius and 5% CO2 .
  • the human wild-type MC4R receptor and 22F plasmid were transfected into HEK293T cells through 1 mg/ml PEI.
  • the cells were evenly spread in a 96-well plate.
  • the ratio of plasmid to PEI was 1:5.
  • the total DNA of salmon sperm cells was used instead of MC4R receptor. Transfected cells served as controls.
  • Figure 5 shows a high-throughput screening platform for GPCR agonists based on the SPS system.
  • a Schematic flow chart of the high-throughput screening platform for GPCR agonists based on the SPS system.
  • the small molecule compound library stored in the form of a 384-well plate is transferred to a sterile 384-well plate through the sonic liquid transfer system Echo550.
  • EBY100-MC4R-SPS yeast is evenly inoculated into the plate containing small molecules and cultured at a constant temperature and humidity of 25 degrees Celsius. , during which the yeast OD 600 changes were measured multiple times with a microplate reader.
  • MC4R agonist screening results Use MATLAB software to calculate and make statistics on the OD 600 of the detected yeast, and draw the yeast growth curves of each well together, in which the growth rate is expressed as the fold change of OD 600 . The highest point is Setmelanotide from the GPCR/G protein compound library.
  • This disclosure uses the EBY100-MC4R-SPS strain to screen a commercial compound library containing approximately 10,000 small molecules. After a period of culture and detection, MC4R agonists included in the GPCR/G protein compound library were screened. Setmelanotide, demonstrated that this system can accurately identify agonists of target GPCRs in a large screening range.
  • this disclosure also successfully identified a drug, Chlorprothixene (trade name: Truxal), which was previously reported to be used to treat mental illness. This drug is an antagonist of dopamine receptors and histamine receptors 24 , which can increase the growth rate of EBY100-MC4R-SPS yeast to a certain extent.
  • Example 10 Detection of the growth rate of recombinant yeast strains EBY100-MC1R-SPS-p2 and EBY100-MC1R-SPS-p3
  • the recombinant plasmids pYD-SPS-MC1R-p2 and pYD-SPS-MC1R-p3 constructed in Example 3 were transferred into Saccharomyces cerevisiae EBY100 in the same manner to obtain Saccharomyces cerevisiae strains EBY100-MC1R-SPS-p2 and EBY100-MC1R- SPS-p3.
  • the final concentrations of the agonist ⁇ -MSH were 1 ⁇ M and 10 ⁇ M, and a tube of yeast with the same volume of sterile water was reserved as a control. Divide yeast from different groups evenly into a sterile 384-well plate, 40 ⁇ l per well, place at 25 degrees Celsius for culture at constant temperature and humidity, and use a microplate reader to measure and calculate the difference in growth rate of yeast in different groups.
  • EBY100-MC1R-SPS-p2 and EBY100-MC1R-SPS-p3 yeast can grow significantly faster than the control group under the action of the exogenous agonist ⁇ -MSH in tryptophan-deficient culture medium, and compared with the agonist concentration of 1 ⁇ M , the recombinant yeast strain under the action of 10 ⁇ M ⁇ -MSH agonist had a significantly increased growth rate in tryptophan-deficient culture medium, and the growth rate was related to the agonist concentration used (Figure 7).
  • Example 11 Detection of the growth rate of recombinant yeast strains EBY100-MC1R-SPS-p2 and EBY100-MC1R-SPS-p3
  • the recombinant plasmid pYD-SPS-MC4R-p2 prepared in Example 4 was transformed into yeast competent cells to obtain the EBY100-SPS-MC4R-p2 strain.
  • Dilute the yeast to OD 600 0.5 using leucine/tryptophan double-deficient medium containing galactose, and divide it evenly into three centrifuge tubes. Add the final concentrations of 1 ⁇ M and 10 ⁇ M to two tubes of yeast respectively.
  • Setmelanotide (SET) the last tube served as a control.
  • Different groups of yeast were cultured at 25 degrees Celsius and 250 rpm with shaking, and a spectrophotometer was used to measure the differences in the growth rates of the yeast in different groups.
  • EBY100-SPS-MC4R-p2 yeast can grow significantly faster than the control group under the action of the exogenous agonist SET in tryptophan-deficient culture medium, and compared with the agonist concentration of 1 ⁇ M, the recombination under the action of 10 ⁇ M SET agonist Yeast strains had significantly increased growth rates in tryptophan-deficient media, and the growth rate correlated with the agonist concentration used (Figure 9).
  • amino acid and nucleotide sequences involved in this disclosure are as follows:
  • Trp1p NTD Amino acid sequence of Trp1p NTD (SEQ ID NO:1):
  • Trp1p CTD Amino acid sequence of Trp1p CTD (SEQ ID NO:3):
  • Amino acid sequence of fusion protein I (based on ⁇ 2AR ) (SEQ ID NO: 10)
  • Amino acid sequence of fusion protein I (based on MC4R) (SEQ ID NO:11)
  • Amino acid sequence of fusion protein I (based on MC1R) (SEQ ID NO:12)
  • Amino acid sequence of fusion protein I (based on MC4R, internal fusion) (SEQ ID NO:13)
  • Amino acid sequence of fusion protein II (fused at site 1) (SEQ ID NO:14)
  • Amino acid sequence of fusion protein II (fused at site 2) (SEQ ID NO:15)
  • Amino acid sequence of fusion protein II (fused at position 3) (SEQ ID NO:16)
  • Nucleotide sequence encoding fusion protein I (based on ⁇ 2AR, first coding region) (SEQ ID NO:18)
  • Nucleotide sequence encoding fusion protein I (based on MC4R, first coding region) (SEQ ID NO:19)
  • Nucleotide sequence encoding fusion protein I (based on MC1R, first coding region) (SEQ ID NO:20)
  • Nucleotide sequence encoding fusion protein I (based on MC4R, internal fusion, first coding region) (SEQ ID NO: 21)
  • Nucleotide sequence encoding fusion protein II (fusion site 1, second coding region) (SEQ ID NO: 22)
  • Nucleotide sequence encoding fusion protein II (fusion site 2, second coding region) (SEQ ID NO: 23)
  • Nucleotide sequence encoding fusion protein II (fusion site 3, second coding region) (SEQ ID NO: 24)
  • Nucleotide sequence encoding fusion protein II (fusion site 4, second coding region) (SEQ ID NO: 25)

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Abstract

La présente divulgation concerne un système de criblage basé sur le stress de survie et un dispositif de criblage à haut débit. En particulier, la présente divulgation concerne une composition de protéines de fusion, des protéines de fusion, une molécule d'acide nucléique recombinante, un vecteur d'expression recombinant, une souche de levure recombinante, un système de criblage basé sur le stress de survie, un dispositif de criblage à haut débit et les utilisations, ainsi qu'un procédé de construction d'une souche de levure recombinante, et un procédé de criblage de médicaments. La protéine de fusion I et la protéine de fusion II fournies par la présente divulgation sont formées par division d'une protéine rapporteur en deux domaines structuraux, puis par fusion de celle-ci avec un RCPG et une protéine G respectivement. Lorsqu'elles sont utilisées pour le criblage de médicaments, la protéine de fusion I et la protéine de fusion II peuvent directement renvoyer des signaux de rapport de survie qui sont générés par combinaison de RCPG activés et de protéines G, ou des signaux de rapport de rétroaction qui sont générés parce que l'effet d'activation des RCPG activés est atténué ou éliminé par compétition, tandis que des processus de régulation de transcription interne et d'amplification en cascade dans la levure ne sont pas nécessaires, et les signaux sont plus réels et crédibles. Par conséquent, les protéines de fusion présentent les avantages d'une haute sensibilité, d'une moindre interférence à partir de signaux non orthogonaux, d'une grande fiabilité et analogues.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005038050A1 (fr) * 2003-10-09 2005-04-28 Ecole Polytechnique Federale De Lausanne Methode d'identification de sites de fragmentation adaptes dans une proteine reporter
US20160124000A1 (en) * 2013-06-10 2016-05-05 The Governing Council Of The University Of Toronto Detection of protein to protein interactions
WO2022129630A1 (fr) * 2020-12-18 2022-06-23 Vrije Universiteit Brussel Peptidomimétiques de protéine g

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005038050A1 (fr) * 2003-10-09 2005-04-28 Ecole Polytechnique Federale De Lausanne Methode d'identification de sites de fragmentation adaptes dans une proteine reporter
US20160124000A1 (en) * 2013-06-10 2016-05-05 The Governing Council Of The University Of Toronto Detection of protein to protein interactions
WO2022129630A1 (fr) * 2020-12-18 2022-06-23 Vrije Universiteit Brussel Peptidomimétiques de protéine g

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HÖRING CARINA, SEIBEL ULLA, TROPMANN KATHARINA, GRÄTZ LUKAS, MÖNNICH DENISE, PITZL SEBASTIAN, BERNHARDT GÜNTHER, POCKES STEFFEN, S: "A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 22, 1 January 2020 (2020-01-01), pages 1 - 18, XP055945700, DOI: 10.3390/ijms21228440 *
TAFELMEYER, P. ; JOHNSSON, N. ; JOHNSSON, K.: "Transforming a (@b/@a)"8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution", CHEMISTRY & BIOLOGY, CURRENT BIOLOGY, LONDON, GB, vol. 11, no. 5, 1 May 2004 (2004-05-01), GB , pages 681 - 689, XP025916620, ISSN: 1074-5521 *

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