Fluorescent probe for screening drug active molecules for treating cancers

Abstract

The invention relates to a fluorescent probe for screening drug active molecules for treating cancers. Specifically, the invention relates to a construction method and application of a fluorescent probe constructed based on an SP / NK-1R mediated signal axis. Specifically, the method comprises the following steps: co-expressing TACR1 (coding NK-1R protein), beta-arrestin-1 / 2 and an ERK kinase active probe; the invention also provides a method for visually detecting the activation or inactivation of the NK-1R protein. The invention also relates to a method for guiding and screening a novel antagonist specifically targeting NK-1R by utilizing visual NK-1R inactivation. And an antagonist which is found by using a visual NK-1R inactivation screening system, is not reported or found yet, can target and block an NK-1R mediated signal channel and inhibits ERK kinase activity probe phase separation.

Description

[0001] This invention claims the priority of a Chinese patent application titled "A Fluorescent Probe Constructed Based on the SP / NK-1R Mediated Signaling Axis" with the application number 2024103071430 and filed on March 18, 2024. The entire content thereof is incorporated herein by reference. Technical Field

[0002] This invention relates to a method for constructing and applying a fluorescent probe based on the SP / NK-1R mediated signaling axis. Specifically, this invention relates to: a fluorescent probe based on the SP / NK-1R mediated signaling axis, which includes co-expressing TACR1, β-arrestin-1 / 2, and an ERK kinase activity probe; a method for visually detecting the activation or inactivation of NK-1R protein; a method for screening novel antagonists specifically targeting NK-1R by using visual NK-1R inactivation guidance; and compounds that have not been reported or discovered by using the visual NK-1R inactivation screening system, which can target and block the NK-1R signaling pathway and inhibit the phase separation of the ERK kinase activity probe. Background Art

[0003] Neurokinin 1 receptor (NK-1R) is widely distributed throughout the nervous system and immune system. Substance P (SP) is its endogenous agonist, and the SP / NK-1R signaling axis is involved in many physiological and pathological processes. In recent years, the SP / NK-1R signaling axis has been considered an independent therapeutic target for cancer treatment. Abnormal expression of NK-1R has been found in various cancer types, including breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, brain cancer, thyroid cancer, and melanoma, etc. SP promotes angiogenesis, proliferation, and metastasis of solid tumor cells in an autocrine, paracrine, or neurosecretory manner. The ERK signaling pathway is closely related to tumor proliferation, migration, and invasion. Many studies have shown that SP-mediated NK-1R activation can cause the activation of ERK1 and ERK2, and this process can be inhibited by pretreatment with NK-1R antagonists.

[0004] TACR1 has different transcripts, and NK-1R exists in two common protein structures. Currently, most theories believe that compared with the full-length NK-1R, the truncated NK-1R has a low affinity for the receptor and a long cell response time, and is usually highly expressed under pathological conditions. In addition, in pharmacological studies, many NK-1R antagonists have shown good selectivity, potency, safety, and bioavailability in preclinical studies and have high research value. However, currently, only three drugs, aprepitant, rolapitant, and netupitant, have been approved by the US Food and Drug Administration for the treatment of nausea and vomiting caused by cancer chemotherapy.

[0005] Develop a fluorescence probe based on the SP / NK-1R-mediated signaling axis, which can observe the activation process of NK-1R in real time, contribute to the in-depth study of the signal transduction mechanism upstream and downstream of NK-1R, and after deepening, the activation of NK-1R under in vivo conditions can be explored.

[0006] Based on the fact that after antagonist pretreatment, SP stimulation cannot cause the activation of NK-1R, resulting in the inability of the ERK kinase activity probe to form fluorescent droplets, there is a significant difference from the formation of fluorescent droplets induced by SP stimulation alone to activate NK-1R. It can be used to guide the development of new antagonists specifically targeting NK-1R, with the advantages of sensitivity, high efficiency, low consumption and greenness, etc., and is a new paradigm for screening drug libraries relying on biological probes.

[0007] The development of fluorescence probes based on NK-1R protein has important scientific significance and clinical application prospects. This will provide new ideas and methods for targeted and individualized treatment of cancer, and contribute to the realization of precision medicine and individualized treatment. Summary of the Invention

[0008] The SP / NK-1R signaling axis is involved in various physiological and pathological regulations, and antagonists of NK-1R have strong application prospects and potential. In order to deepen the mechanism of action of NK-1R in tumorigenesis and development, in order to visualize the activation of NK-1R in tumor cells, and in order to develop new antagonists specifically targeting NK-1R, the inventor has developed a fluorescence probe constructed based on the SP / NK-1R-mediated signaling axis.

[0009] Therefore, on the one hand, the present invention provides a biological probe, the components of which include two parts: a probe composed of the NK-1R signaling pathway and a probe for detecting ERK kinase activity. The probe composed of the NK-1R signaling pathway, TACR1 and β-arrestin-1 or TACR1 and β-arrestin-2, are independently expressed plasmids; the probe for detecting ERK kinase activity is a fusion protein, or a conjugate of ERK-sub and a fluorescent compound and a conjugate of WW and a fluorescent compound.

[0010] Preferably, in the present invention, the NK-1R and β-arrestin-1 / 2 proteins, ERK-sub and WW polypeptides are derived from mammals.

[0011] More preferably, in the present invention, the NK-1R and β-arrestin-1 / 2 proteins, ERK-sub and WW polypeptides are derived from humans, and they have the following amino acid sequences, or are modified on the basis of the following amino acid sequences:

[0012] TACR1:

[0013] MDNVLPVDSDLSPNISTNTSEPNQFVQPAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTETMPSRVVCMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITLWASEIPGDSSDRYHEQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAGDYEGLEMKSTRYLQTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTMTESFSFSSNVLS;

[0014] ERK-sub:

[0015] MADQLTEEWSTVPRTGPDVPRTPVGKAKLSFQFPGG;

[0016] WW:

[0017] MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSSGGKNGQGEPAR。

[0018] β-arrestin-1:

[0019] MGDKGTRVFKKASPNGKLTVYLGKRDFVDHIDLVDPVDGVVLVDPEYLKERRVYVTLTCAFRYGREDLDVLGLTFRKDLFVANVQSFPPAPEDKKPLTRLQERLIKKLGEHAYPFTFEIPPNLPCSVTLQPGPEDTGKACGVDYEVKAFCAENLEEKIHKRNSVRLVIRKVQYAPERPGPQPTAETTRQFLMSDKPLHLEASLDKEIYYHGEPISVNVHVTNNTNKTVKKIKISVRQYADICLFNTAQYKCPVAMEEADDTVAPSSTFCKVYTLTPFLANNREKRGLALDGKLKHEDTNLASSTLLREGANREILGIIVSYKVKVKLVVSRGGLLGDLASSDVAVELPFTLMHPKPKEEPPHREVPENETPVDTNLIELDTNDDDIVFEDFARQRLKGMKDDKEEEEDGTGSPQLNNR;

[0020] β-arrestin-2:

[0021] MGEKPGTRVFKKSSPNCKLTVYLGKRDFVDHLDKVDPVDGVVLVDPDYLKDRKVFVTLTCAFRYGREDLDVLGLSFRKDLFIATYQAFPPVPNPPRPPTRLQDRLLRKLGQHAHPFFFTIPQNLPCSVTLQPGPEDTGKACGVDFEIRAFCAKSLEEKSHKRNSVRLVIRKVQFAPEKPGPQPSAETTRHFLMSDRSLHLEASLDKELYYHGEPLNVNVHVTNNSTKTVKKIKVSVRQYADICLFSTAQYKCPVAQLEQDDQVSPSSTFCKVYTITPLLSDNREKRGLALDGKLKHEDTNLASSTIVKEGANKEVLGILVSYRVKVKLVVSRGGDVSVELPFVLMHPKPHDHIPLPRPQSAAPETDVPVDTNLIEFDTNYATDDDIVFEDFARLRLKGMKDDDYDDQLC。

[0022] For the modification of this amino acid sequence, various modifications and changes can be made without affecting its function to meet different experimental requirements or enhance its specific functions. The following are possible modification methods:

[0023] Point mutation: Alter the specific structure or function of NK-1R and β-arrestin-1 / 2 proteins, ERK-sub, and WW polypeptides by substitution, insertion, or deletion of a single amino acid. For example, mutations can be used to enhance the stability of fluorescent probes or the intensity of fluorescent signals.

[0024] Insertion of functional tags: In addition to linker peptides and fluorescent proteins, other functional tags such as His-Tag, HA-Tag, Flag-Tag, etc. can be inserted into NK-1R and β-arrestin-1 / 2 proteins, ERK-sub, and WW polypeptides for further experimental operations or to improve the detection sensitivity of the probes.

[0025] Deletion or replacement of domains: Specific domains in NK-1R and β-arrestin-1 / 2 proteins, ERK-sub, and WW polypeptides can be deleted or replaced to regulate their interactions with other molecules or to improve their stability and activity in cells.

[0026] Chemical modification: Chemical modification groups such as phosphorylation, methylation, or acylation are introduced at specific residues of NK-1R and β-arrestin-1 / 2 proteins, ERK-sub, and WW polypeptides by chemical methods to regulate their structure and function.

[0027] Preferably, in the present invention, the linker peptides between ERK-sub, EGFP or WW and EGFP, EGFP and HT4, and EGFP and HT6 are flexible linker peptides. More preferably, the linker peptides include glycine and / or serine.

[0028] Preferably, in the present invention, the linker peptide between ERK-sub-EGFP-HT6 and WW-EGFP-HT4 is a self-cleaving peptide. More preferably, the linker peptide is T2A.

[0029] Preferably, in the present invention, the fluorescent protein is blue fluorescent protein, green fluorescent protein, red fluorescent protein, or yellow fluorescent protein.

[0030] Preferably, in the present invention, the blue fluorescent protein includes but is not limited to BFP, EBFP, TagBFP, mTagBFP2, CyPet, Cerulean, mTurquoise.

[0031] Preferably, in the present invention, the green fluorescent protein includes but is not limited to GFP, EGFP, mEGFP, mWasabi, ZsGreen, mNeonGreen.

[0032] Preferably, in the present invention, the red fluorescent protein includes, but is not limited to, TagRFP, DsRed, mCherry, mStrawberry, dTomato, tdTomato, mApple, mKate2.

[0033] Preferably, in the present invention, the yellow fluorescent protein includes, but is not limited to, YFP, EYFP, Venus, mVenus, Citrine, YPet.

[0034] Preferably, in the present invention, the fluorescent compound includes, but is not limited to, FITC (fluorescein isothiocyanate), FAM (carboxyfluorescein), TET (tetrachlorofluorescein), TRITC (tetramethylrhodamine isothiocyanate), TAMRA (carboxytetramethylrhodamine), cyanine dyes CY3, CY5, CY7.

[0035] In the present invention, the modification of the ANXA1 protein, in addition to inserting the above-mentioned linker peptide, fluorescent protein or fluorescent compound, also includes inserting other functional modification tags, including, but not limited to, His-Tag, HA-Tag, Flag-Tag, GST-Tag, Myc-Tag, MBP-Tag (Maltose binding protein tag), HOTag (Homo-oligomerictag).

[0036] Another aspect of the present invention provides the use of visualizing the phase transition degree of the ERK kinase activity probe to characterize NK-1R activation. By using a fluorescent probe constructed based on the SP / NK-1R signaling axis, the activation level of the NK-1R protein can be converted into a biomolecular phase separation signal, thereby realizing rapid, real-time and efficient monitoring of the activation level of the NK-1R protein, and can be used to guide the screening of novel antagonists specifically targeting NK-1R.

[0037] Specifically, the fluorescent probe of the present invention can achieve the following functions:

[0038] Quantitative analysis: The degree of phase transition of the fluorescent probe is positively correlated with the activation of the NK-1R protein in the cell. By measuring the number and intensity of the green fluorescent droplets undergoing phase transition, the activation of ERK1 / 2 in the cell can be accurately quantitatively analyzed, thereby evaluating the activation level of the NK-1R protein in different cell states or treatment conditions.

[0039] Drug screening: In combination with the ERK kinase activity probe, it can visually detect the inactivation of the activated NK-1R under the stimulation of a clinical antagonist, resulting in the disappearance of the fluorescent droplets formed by phase separation. This process can be used to guide the screening of novel antagonists specifically targeting NK-1R.

[0040] Preferably, in the present invention, the novel antagonist refers to a drug that has not been reported or discovered, can target and block the NK-1R signaling pathway, and inhibit the phase separation of the ERK kinase activity probe, including traditional Chinese medicines, chemical drugs, and biological drugs. Compared with traditional drug screening methods, the biological probe constructed based on the SP / NK-1R axis pathway has the advantages of being environmentally friendly, cost-effective, and easy to operate; at the same time, due to the principle of the probe combining with phase separation, compared with traditional chemiluminescence or biological probes, this probe has the advantages of a large dynamic range change (appearance and disappearance of fluorescent droplets), reversibility, and low toxicity.

[0041] In the present invention, for the method of expressing the fluorescent probe in cells, pcDNA3.1-TACR1, pcDNA3.1-ERK-EGFP-HT6-T2A-WW-EGFP-HT4, and pcDNA3.1-β-arrestin-1 / 2 compound conjugates are directly co-transfected into cell culture medium using calcium phosphate transfection technology for the synthesized conjugates, or the conjugates are co-incubated with liposomes, or the cell membrane is perforated by electric stimulation to enable them to enter the cells.

[0042] In the present invention, the transfection reagents include but are not limited to calcium phosphate transfection reagents, liposomes, and linear polyethyleneimine (PEI).

[0043] Preferably, in the present invention, for the calcium phosphate transfection technology, 6 μL of HEPES buffer (HBS), 0.35 μL of calcium chloride (CaCl2), and a total plasmid amount of 250 ng are required per single well.

[0044] In the present invention, the method for measuring the fluorescence signal includes taking fluorescence images using a fluorescence microscope and detecting the fluorescence signal intensity using a microplate reader with a fluorescence module. For the use of the fluorescence microscope, a fixed excitation light intensity and exposure time for shooting are set, and fluorescence images of candidate compound-treated and untreated samples are taken.

[0045] Preferably, in the present invention, the fluorescence microscope is a high-content imaging system, and a high-content analysis system is used for the analysis of fluorescence images. Description of the Drawings

[0046] Figure 1 According to the CDS sequence of TACR1 and the multiple cloning sites of the pcDNA3.1 expression vector, a map of the pcDNA3.1-TACR1 expression vector to be constructed is designed.

[0047] Figure 2Based on the CDS sequences of β-arrestin-1 / 2 and the multiple cloning sites of the pcDNA3.1 expression vector, the map of the pcDNA3.1-β-arrestin-1 / 2 expression vector to be constructed was designed.

[0048] Figure 3 Based on the CDS sequences of the ERK specific substrate domain, EGFP, HT6, T2A, WW domain, HT4 and the multiple cloning sites of the pcDNA3.1 expression vector, the map of the pcDNA3.1-ERK-sub-EGFP-HT6-T2A-WW-EGFP-HT4 expression vector to be constructed was designed.

[0049] Figure 4 Schematic diagram of a related fluorescent probe constructed based on the SP / NK-1R-mediated signal axis. When only SP is present, NK-1R is activated and mediates downstream signal cascades, releasing Ca 2+ signal, which causes ERK activation. The activated ERK will specifically recognize and phosphorylate its specific substrate domain. The activated specific substrate domain is recognized and bound by the downstream WW domain-containing fragment. Under the action of the self-oligomerizing short peptides HT6 and HT4, protein aggregation occurs and a phase transition occurs to form fluorescent droplets; when pretreated with aprepitant, even if SP is present, the NK-1R downstream signaling pathway cannot be activated, so no phase transition occurs; when there are no endogenous ligands such as SP or exogenous agonists, the NK-1R downstream signaling pathway cannot be activated, and no phase transition occurs either.

[0050] Figure 5 For the co-transfection experiment in 293T cells. In 293T cells, the ERK kinase activity probe was co-transfected with pcDNA3.1 empty vector, TACR1, β-arrestin-1 / 2 according to different combinations, and three stimuli, namely PBS, SP, and aprepitant + SP, were used to simulate three physiological and pathological conditions of non-activation, activation, and inhibitory inactivation. It can be found that only when stimulated by SP, the ERK kinase activity probe undergoes a phase transition; when treated with PBS or pretreated with aprepitant, no phase transition occurs. When SP stimulation is satisfied, the presence of β-arrestin-1 / 2 can significantly reduce the activation of the background level of the ERK kinase activity probe. In comparison, β-arrestin-1 has a lower activation background of the kinase activity probe.

[0051] Under the stimulation of SP alone, the ERK kinase activity probe undergoes a phase transition only under the condition that NK-1R (kinase) is present; when NK-1R is absent, no phase transition occurs. Specific implementation methods

[0052] The principle of the fluorescent probe constructed based on the SP / NK-1R-mediated signal axis of the present invention is as follows: The upstream NK-1R binds to an agonist or an endogenous ligand. Due to the signal cascade reaction, ERK activation is induced. The activated ERK will specifically recognize and bind to its specific substrate domain, causing phosphorylation of the Thr site. The phosphorylated specific substrate domain is recognized and bound by the downstream WW domain-containing fragment. That is, after the phosphorylation site of the ERK specific substrate domain is activated, it interacts with the WW domain, and protein aggregation and phase transition occur to form fluorescent droplets under the action of the spontaneously oligomerizing peptides HT6 and HT4. This process can be inhibited by pretreatment with an NK-1R antagonist, so it can be used for screening NK-1R specific antagonists.

[0053] For the fluorescent probe constructed based on the SP / NK-1R-mediated signal axis of the present invention, fluorescence labeling of the ERK kinase activity probe can be achieved by including eukaryotic expression systems, prokaryotic expression systems, or chemical synthesis.

[0054] For the NK-1R and proteins of the present invention, their sources can be various animals such as humans, mice, rats, chickens, pigs, dogs, cows, monkeys, chimpanzees, horses, rabbits, elephants, etc., and can be modified on the basis of their amino acid sequences, such as amino acid insertion, substitution, or deletion.

[0055] In a specific embodiment of the present invention, the specific amino acid sequence of the NK-1R protein derived from humans is:

[0056] MDNVLPVDSDLSPNISTNTSEPNQFVQPAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTETMPSRVVCMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITLWASEIPGDSSDRYHEQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAGDYEGLEMKSTRYLQTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTMTESFSFSSNVLS.

[0057] Alternatively, more preferably, the NK-1R protein is a singly or doubly mutated protein. Exemplarily, the amino acid sequence of the doubly mutated NK-1R protein is as follows:

[0058] MDNVLPVDSDLSPNISTNTSEPNQFVQPAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAHMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTETMPSRVVCMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITLWASEIPGDSSDRYHEQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAGDYEGLEMKSTRYLQTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTMTESFSFSSNVLS.

[0059] For the linker peptide of the present invention, it can be a flexible linker peptide or a rigid linker peptide, connecting ERK-sub to EGFP, WW to EGFP, EGFP to Hotag4 (HT4), and EGFP to Hotag6 (HT6). Meanwhile, since ERK and WW need to specifically recognize each other rather than being tandemly expressed, an in vivo cleavable linker peptide needs to be added to form independent ERK-sub hexamers and WW tetramers.

[0060] In a specific embodiment of the present invention, the specific amino acid sequence of the linker peptide is:

[0061] EGRGSLLTCGDVEENPGP (2A self-cleaving peptide)

[0062] GSAGGSAGGSAGGSAGGSAGGSAGGSAGG (linker peptide between EGFP and Hotag4 / 6)

[0063] TCCGGACTCAGATCTGGCAGCGGTGGAGGC (linker peptide between ERK-sub and EGFP or between WW and EGFP)

[0064] For the fluorescent protein of the present invention, it can be any protein that can emit fluorescence and is used to label the ANXA1 protein.

[0065] In a specific embodiment of the present invention, the specific amino acid sequence of green fluorescent protein EGFP is: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0066] For the fluorescent compound of the present invention, it can be any compound capable of emitting fluorescence and is used to label ERK-sub and WW, including but not limited to FITC (fluorescein isothiocyanate), FAM (carboxyfluorescein), TET (tetrachlorofluorescein), TRITC (tetramethylrhodamine isothiocyanate), TAMRA (carboxytetramethylrhodamine), cyanine dyes CY3, CY5, CY7.

[0067] For the fluorescent probe of the present invention, in addition to inserting the above-mentioned linker peptide, fluorescent protein or fluorescent compound into the modification of ERK-sub and WW, it also includes inserting other functional modification tags, including but not limited to His-Tag, HA-Tag, Flag-Tag, GST-Tag, Myc-Tag, MBP-Tag, HO-Tag.

[0068] In fact, the present invention provides great advantages for the selection of specific proteins or linkers, although some elements are known in the prior art. To better understand the content of the present invention, the content of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0069] Example

[0070] 1. Construction of fluorescent probe expression vector

[0071] 1.1 Primer design

[0072] According to the CDS sequence of TACR1 and the multiple cloning sites of the pcDNA3.1 expression vector, the map of the pcDNA3.1-TACR1 expression vector to be constructed was designed ( Figure 1 ).

[0073] According to the CDS sequences of β-arrestin-1 / 2 and the multiple cloning sites of the pcDNA3.1 expression vector, the map of the pcDNA3.1-β-arrestin-1 / 2 expression vector to be constructed was designed ( Figure 2 ).

[0074] According to the CDS sequences of the ERK specific substrate domain (including the peptide segment derived from CDC25C containing the Thr phosphorylation site and the ERK kinase specific recognition peptide segment), EGFP, HT6, T2A, WW polypeptide fragment, HT4 and the multiple cloning sites of the pcDNA3.1 expression vector, the map of the pcDNA3.1-ERK-sub-EGFP-HT6-T2A-WW-EGFP-HT4 expression vector to be constructed was designed ( Figure 3 ).

[0075] Based on this, specific PCR primers were designed:

[0076] TACR1-F:

[0077] 5’ACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGATAACGTCCTCCCGGTG3’;

[0078] TACR1-R:

[0079] 5’TCAGCGGGTTTAAACGGGCCCGAATTCTTAGGAGAGCACATTGGAGGA3’;

[0080] β-arrestin-1-F:

[0081] 5’ACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGGCGACAAAGGGACCCGA3’;

[0082] β-arrestin-1-R:

[0083] 5’TCAGCGGGTTTAAACGGGCCCGAATTCTTATCTGTTGTTGAGCTGTGG3’;

[0084] β-arrestin-2-F:

[0085] 5’ACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGGGGAGAAACCCGGGACC3’;

[0086] β-arrestin-2-R:

[0087] 5’TCAGCGGGTTTAAACGGGCCCGAATTCTTAGCAGAGTTGATCATCATA3’;

[0088] ERK-sub-F:

[0089] 5’ACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGCTGACCAACTGACTGAA3’;

[0090] ERK-sub-R:

[0091] 5’GCTGCCAGATCTGAGTCCGGACTCGAGTCCGCCCGGGAATTGGAATGA3’;

[0092] EGFP-HT4 / 6-F:

[0093] 5’AGATCTGGCAGCGGTGGAGGCGATATCATGGTGAGCAAGGGCGAGGAG3’;

[0094] EGFP-HT4-R:

[0095] 5’TCAGCGGGTTTAAACGGGCCCGAATTCTTAGATTTTCTTCAGCCACTT3’;

[0096] EGFP-HT6-R:

[0097] 5’TAGCAGACTTCCTCTGCCCTCGGTACCCCCTTTGAGGGACTTGGCAAT3’;

[0098] T2A-F:

[0099] 5’ATTGCCAAGTCCCTCAAAGGGGGTACCGAGGGCAGAGGAAGTCTGCTA3’;

[0100] T2A-R:

[0101] 5’CAGCTTCTCCTCGTCCGCCATGGTGGCAAGCTTTGGGCCAGGATTCTCCTCGAC3’;

[0102] WW-F:

[0103] 5’GTCGAGGAGAATCCTGGCCCAAAGCTTGCCACCATGGCGGACGAGGAGAAGCTG3’;

[0104] WW-R:

[0105] 5’GCTGCCAGATCTGAGTCCGGACTCGAGCCTGGCAGGCTCCCCCTGCCC3’;

[0106] Among them, the primers TACR1-F, β-arrestin-1-F, β-arrestin-2-F, ERK-sub-F, and WW-F contain the NheⅠ restriction enzyme site; the primers ERK-sub-R and WW-R contain the Xhol I restriction enzyme site; the primer EGFP-HT4 / 6-F contains the ECOR V restriction enzyme site; the primers T2A-F and EGFP-HT6-R contain the Kpn I restriction enzyme site; the primers contain the HondⅢ restriction enzyme site; the primers TACR1-R, β-arrestin-1-R, β-arrestin-2-R, and EGFP-HT4-R contain the ECOR I restriction enzyme site.

[0107] 1.2 Cloning DNA Fragments

[0108] Set up the following reaction system in a 50 μL PCR reaction tube:

[0109]

[0110] The reaction conditions are as follows:

[0111]

[0112] Perform PCR reaction according to the reaction system and reaction conditions in the above table to amplify the DNA fragment.

[0113] 1.3 Enzyme Digestion of Vector

[0114] Set up the following enzyme digestion reaction system for the pcDNA3.1 empty plasmid:

[0115]

[0116] After reacting at 37°C for 1 hour, terminate the reaction. After the PCR reaction product and the enzyme digestion product, perform 1% agarose gel electrophoresis identification after the reaction, and cut and recover the target fragment.

[0117] 1.4 Construction of Recombinant Vector

[0118] After the enzyme-digested DNA fragment is purified by cutting the gel, according to the instructions of the Vazyme ClonExpress II (Cat#C112) and ClonExpress MultiS (Cat#C113) one-step cloning kits, the DNA fragment is ligated with the vector to form a recombinant vector:

[0119] 1.5 Transformation of the ligation product

[0120] Add all 10 μL of the ligation product to 100 μL of DH5α competent cells, and place on ice for 1 hour; heat shock at 42 °C for 90 seconds, and quickly place on ice for 5 minutes; add 600 μL of LB culture medium preheated to 37 °C; shake at 37 °C and 220 rpm for 1 hour, centrifuge and spread all on an LB plate containing 100 μg / ml Amp, and incubate inverted at 37 °C overnight.

[0121] 1.6 Identification of positive clones

[0122] Randomly pick 4 monoclonal colonies into a test tube containing 3 mL of LB culture medium with 100 μg / mL Amp, shake at 37 °C and 220 rpm for 4 hours, take 100 μL for centrifugation, take the bacterial cell pellet, resuspend it with 50 μL of ddH2O, boil in a water bath for 5 minutes, centrifuge and take 1 μL of the supernatant as a template for PCR identification.

[0123] Set up the following reaction system in a 10 μL PCR reaction tube:

[0124]

[0125] The reaction conditions are as follows:

[0126]

[0127] After the reaction, identify the PCR products by 1% agarose gel electrophoresis.

[0128] 1.7 Plasmid extraction and DNA sequencing

[0129] Continue to culture the bacterial solution identified as positive by colony PCR, extract the plasmid and send it for sequencing, and screen to obtain the pcDNA3.1-TACR1, pcDNA3.1-β-arrestin-1 / 2, and pcDNA3.1-ERK-sub-EGFP-HT6-T2A-WW-EGFP-HT4 expression vectors with correct sequences.

[0130] 2. Fluorescent probe expression

[0131] 2.1 Cell plating

[0132] The 293T cells in the logarithmic growth phase were digested with trypsin, resuspended in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (Fetal Bovine Serum), and collected in a centrifuge tube. The cells were centrifuged at 700 rpm for 5 minutes and washed with PBS (Phosphate-Buffered Saline). After repeating this 2 times, the cell pellet was resuspended in DMEM medium containing 10% FBS.

[0133] The cell density was calculated using a cell counter, and the cells were plated at 20,000 cells per well. After 6 - 8 hours, the 293T cells adhered to the plate. The 96-well plates were grouped into six groups: pcDNA3.1 empty vector + ERK kinase activity probe, TACR1 + ERK kinase activity probe, pcDNA3.1 empty vector + ERK kinase activity probe + β-arrestin-1, TACR1 + ERK kinase activity probe + β-arrestin-1, pcDNA3.1 empty vector + ERK kinase activity probe + β-arrestin-2, and TACR1 + ERK kinase activity probe + β-arrestin-2.

[0134] 2.2 Preparation of plasmid-calcium phosphate transfection reagent mixed solution

[0135] According to the above grouping, the ERK kinase activity probe was mixed with pcDNA3.1 empty vector, TACR1, and β-arrestin-1 / 2. The total amount of plasmid per well was 250 ng, TACR1 was 100 ng, β-arrestin-1 / 2 was 100 ng, and the ERK kinase activity probe was 50 ng. The wells with less than 250 ng of plasmid were supplemented with pcDNA3.1 empty vector.

[0136] According to the transfection conditions in the 96-well cell culture plate, 6 μL of 1×HBS buffer, 0.35 μL of 2.5 M CaCl2 solution, and a total of 250 ng of plasmid were thoroughly mixed in an eight-well strip, allowed to stand for 20 minutes, and then added dropwise to the cell culture dish.

[0137] 2.3 Drug treatment

[0138] Sixteen hours after transfection, PBS buffer or different concentrations of the NK-1R antagonist aprepitant were pre-added to the 96-well plates in different groups and incubated for 30 minutes.

[0139] Subsequently, 100 nM SP was added and incubated for 1 hour.

[0140] 3. Observation under a fluorescence microscope

[0141] Images were recorded using high-content imaging, as Figure 5As shown, the changes in EGFP green fluorescence were observed. No green fluorescent droplets were formed in the blank control with PBS added and the negative control without SP stimulation, while green fluorescent droplets were formed in the positive control with SP stimulation. No fluorescent droplets were formed under SP stimulation after pretreatment with the antagonist aprepitant. This indicates that the probe has characteristics such as a large dynamic range (appearance and disappearance of fluorescent droplets) and fast kinetics.

[0142] The activation level of NK-1R in living cells was quantified using the green fluorescence intensity formed by each cell.

[0143] The cells were cultured for an additional 24 hours. Under a fluorescence microscope, the green fluorescent droplets formed in the positive control group disappeared, and no obvious changes in cell morphology were observed, indicating advantages such as reversibility and low toxicity in this process.

[0144] Those of ordinary skill in the art should understand that the above description is only the specific implementation manner of the present invention, not all implementation manners. Methods and raw materials other than those specifically exemplified can be adopted in the practice of the present invention without undue experimentation. All known functional equivalents of any such methods and raw materials are expected to be included in the present invention. Those skilled in the art should also understand that various changes and modifications can be made to the present invention described in this specification and the claims, and the present invention includes all such changes and modifications.

Claims

1. A fluorescence probe system constructed based on the SP / NK-1R-mediated signal axis, the fluorescence probe system comprising: The NK-1R signaling pathway component probe and the ERK kinase activity detection probe, which comprise or consist of TACR1 and β-arrestin-1 or TACR1 and β-arrestin-2, and the TACR1 and β -arrestin-1 or TACR1 and β-arrestin-2 are respectively encoded by independently expressed plasmids; The ERK kinase activity detection probe, which is a fusion protein, including an ERK-specific substrate domain (ERK-sub), a WW domain (WW), a fluorescent protein and a short hydrophobic amino acid peptide; Wherein, ERK-sub is connected to the fluorescent protein, WW is connected to the fluorescent protein, and the fluorescent protein is respectively connected to HOTag6 (HT6) and HOTag4 (HT4), and the structures are respectively ERK-sub-EGFP-HOTag6 and WW-EGFP-HOTag4.

2. The fluorescent probe according to claim 1, wherein The linker peptide between the ERK-sub-EGFP-HOTag6 and WW-EGFP-HOTag4 is the T2A self-cleaving peptide.

3. The fluorescent probe according to claim 1, wherein The ERK-sub consists of a peptide segment derived from CDC25C containing a phosphorylation site and an ERK kinase-specific recognition peptide segment.

4. The fluorescent probe according to claim 1, wherein The ERK-sub, WW, TACR1, β-arrestin-1 or 2 are derived from mammals, preferably humans.

5. The fluorescence probe according to claim 1, wherein the linker peptide between the ERK-sub, WW, EGFP-HOTag6 and EGFP-HOTag4 is a flexible linker peptide, and the flexible linker peptide contains glycine and / or serine.

6. The fluorescence probe according to claim 1, wherein the fluorescent protein is a blue fluorescent protein, a green fluorescent protein, a red fluorescent protein or a yellow fluorescent protein.

7. The fluorescence probe according to claim 1, wherein the fluorescent protein is functionally replaced with a fluorescent compound, and is selected from FITC (fluorescein isothiocyanate), FAM (carboxyfluorescein), TET (tetrachlorofluorescein), TRITC (tetramethylrhodamine isothiocyanate), TAMRA (carboxytetramethylrhodamine), CY3, CY5 or CY7.

8. A method for screening a novel antagonist specifically targeting NK-1R using the fluorescence probe according to claim 1, comprising: (1) Expressing the fluorescence probe in cells; (2) Adding a candidate compound to the cell culture medium; (3) Inducing NK-1R activation by SP stimulation; (4) Evaluating the inhibitory effect of the candidate compound on the NK-1R-mediated signaling pathway by the formation or disappearance of fluorescent droplets.

9. The method according to claim 8, wherein the fluorescence probe is used for real-time monitoring of the NK-1R activation level and for screening of NK-1R-specific antagonists.

10. A drug screening method, comprising using the fluorescence probe according to claim 1 to screen for antagonists capable of targeting and blocking the NK-1R signaling pathway.

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