Selective modulators against human adrenergic receptor alpha2A and muscarinic acetylcholine receptor M4

Abstract

The present invention relates to a plurality of engineered variants based on muscarinic toxin MT3 which specifically bind to muscarinic acetylcholine receptor M4 or adrenergic receptor alpha 2A, preferably human muscarinic acetylcholine receptor M4 or human adrenergic receptor alpha 2A.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a specific targeting human adrenergic receptor or muscarinic acetylcholine receptor M4 selective regulation molecule and use thereof. BACKGROUND

[0002] Adrenergic receptors (AR) and muscarinic acetylcholine receptors (mAChR) are two important members of the G protein coupled receptor (GPCR) family, and play an indispensable role in regulating the physiological functions of the body. Among them, adrenergic receptors can be divided into three subfamilies, each of which contains three members (α1A / B / D, α2A / B / C and β1 / 2 / 3); and the muscarinic acetylcholine receptor family contains five subfamily members (M1AChR~M5AChR), each of which has different distribution and functional characteristics in the human body. For example, the α2A receptor in the adrenergic receptor family is mainly expressed in the central nervous system and peripheral tissues, and is involved in the fine regulation of neurotransmission and cardiovascular function. Blocking the signal pathway of the α2A receptor can reduce the inflammatory response of sepsis and reverse pain and morphine analgesia; while the α2B receptor is widely distributed in blood vessels, and activation of the α2B receptor will cause the body's blood pressure to rise. In the muscarinic acetylcholine receptor family, M4AChR has a high expression level in specific areas of the central nervous system, especially in the key brain areas of the forebrain and striatum of the brain, and plays an important role in the process of learning and memory. Agonists or antagonists targeting M4AChR have potential therapeutic effects in the treatment of central nervous system diseases (such as Alzheimer's disease, Parkinson's disease, etc.).

[0003] At present, a variety of drugs have been developed for AR and mAChR and applied in clinical. For example, the agonists of the α2AR subfamily, clonidine and methyldopa, show efficacy in the treatment of hypertension and certain types of arrhythmia; and mAChR agonists or antagonists such as bethanechol, oxybutynin and atropine have been widely used in the treatment of gastrointestinal, urinary system and ophthalmic diseases. However, these drugs usually lack strict subtype selectivity, resulting in their side effects in the treatment of diseases related to α 2AAR or M4AChR subtypes, and also interacts with other subtypes of receptors, thereby causing non-specific pharmacological effects, such as dry mouth, blurred vision, palpitations, constipation, and cognitive impairment, etc. These side effects not only limit the dosage and long-term application of the drug, but also affect the therapeutic effect and the quality of life of patients. Therefore, developing drugs with high subtype selectivity to reduce side effects in non-target tissues and improve therapeutic effect is still an important goal of pharmacological research. However, due to the high sequence homology and structural similarity among members of the AR or mAChR family, especially the high conservation in the orthosteric binding site region, it has been a great challenge to develop subtype-selective ligands.

[0004] Compared with small molecule ligands, protein ligands can usually occupy a larger binding surface on the target receptor, thereby interacting with non-conserved regions of the target receptor to achieve high selective recognition of specific receptor subtypes. This provides new possibilities for specific drug development. The toxin protein MT3 (Muscarinic toxin 3) isolated from mamba venom has been shown to exhibit high affinity for a variety of G protein-coupled receptors (GPCRs), including α2A adrenergic receptor (α2AAR) and M4 muscarinic acetylcholine receptor (M4AChR). 2A In particular, in the adrenergic receptor family, MT3 can interact with multiple subtypes (such as α1A, α1B, α1D, α2A and α2C) with similar high affinity, with a pKi value of about 7.5; while in the muscarinic acetylcholine receptor family, the affinity of MT3 for M4 has a pKi value of about 9, which is significantly higher than that for M1, with a difference of at least two orders of magnitude.

[0005] In addition, MT3 has a unique "three-finger folding" structure, which is composed of five β-folding sheets and a "finger" (Finger) of a flexible peptide segment, and a "finger loop" (Finger loop) of three loop regions, which together participate in the interaction with the receptor. The high affinity binding property of MT3 and its structural plasticity make it an ideal candidate for protein engineering. By engineering the receptor binding region of MT3, it is hoped to design subtype-specific modulators for different receptor subtypes (such as α2A and M4), which can help to fill the gap in the research of subtype-specific ligands for α2A and M4, and provide a new strategy and research direction for related drug development and disease treatment. SUMMARY

[0006] The present application provides a yeast surface display screening method, which uses toxin protein MT3 as the core skeleton to direct the modification of the receptor binding region of MT3, and successfully develops a variety of subtype-specific modulators for adrenergic receptors α2A (α2AAR) and M4 muscarinic acetylcholine receptors (M4AChR) by engineering the receptor binding region of MT3. 2AAR) (hereinafter referred to as α2A) and muscarinic acetylcholine receptor M4 (M4AChR) (hereinafter referred to as M4). Through functional experiments, the regulatory molecules of the present application can effectively regulate the signal transduction pathway mediated by specific receptors, and have high subtype selectivity.

[0007] Firstly, the present application obtains specific regulatory molecules for adrenaline receptor α2A through screening. These molecules can specifically block the signal pathway activation induced by α2A agonists, have potential application prospects, and can be used for the treatment of α2A related diseases. Secondly, the present application also screens specific regulatory molecules for muscarinic acetylcholine receptor M4, which can effectively block the signal transduction mediated by M4 agonists, and provides a new treatment method for M4 related diseases. The present application provides a new tool and platform for the development of α2A and M4 specific drugs, which has important application value and potential in the field of disease treatment, and can also be used for related basic research.

[0008] Specifically, the present application provides the following technical solutions:

[0009] On the one hand, the present application provides a variety of adrenaline receptor specific regulatory molecules for adrenaline receptor α2A, which can specifically bind to adrenaline receptor α2A, preferably human adrenaline receptor α2A. Among them, the adrenaline receptor specific regulatory molecule based on wild type MT3 (SEQ ID NO: 1) comprises one or more substitution mutations selected from the group consisting of H29T, H29E, H29S, Y30P, Y30S, Y30H, V31I, V31Y, T36V, E37R, Y52G, D53E, S54T, S54I, S54Y, S54R, S54L, S54T.

[0010] In one embodiment, the combination of substitution mutations comprises a combination of H29T, Y30P, T36V, Y52G, S54I substitution mutations (A64, SEQ ID NO: 2). In one embodiment, the combination of substitution mutations comprises a combination of H29T, Y30S, V31I, T36V, Y52G, S54Y substitution mutations (A66, SEQ ID NO: 3). In one embodiment, the combination of substitution mutations comprises a combination of H29E, V31Y, T36V, E37R, Y52G, S54Y substitution mutations (RD7, SEQ ID NO: 4). In one embodiment, the combination of substitution mutations comprises a combination of H29S, Y30H, T36V, Y52G, S54R substitution mutations (A2S5, SEQ ID NO: 5). In one embodiment, the combination of substitution mutations comprises a combination of Y52G, S54R substitution mutations (A2-3, SEQ ID NO: 6). In one embodiment, the combination of substitution mutations comprises a combination of Y52G, S54L substitution mutations (A2-6, SEQ ID NO: 7). In one embodiment, the combination of substitution mutations comprises a combination of Y52G, D53E, S54T substitution mutations (A2-10, SEQ ID NO: 8). In some embodiments, the combination of substitution mutations comprises, consists or consists essentially of a sequence as set forth in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, a sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more, preferably 2 or 3, conservative amino acid mutations, preferably substitutions, insertions or deletions, compared to said sequence.

[0011] In another aspect, the present application provides a muscarinic acetylcholine receptor specific modulator against muscarinic acetylcholine receptor M4, which is capable of specifically binding to muscarinic acetylcholine receptor M4, preferably to human muscarinic acetylcholine receptor M4, wherein said muscarinic acetylcholine receptor specific modulator comprises one or more substitution mutations based on wild type MT3 (SEQ ID NO: 1), said substitution mutations being selected from the group consisting of N7T, T8G, T8S, I9L, Y30A, Y30S, T36V, T36A, E37R.

[0012] In one embodiment, the combination of substitution mutations comprises a combination of Y30A, T36V, E37R substitution mutations (M4S3, SEQ ID NO: 9). In one embodiment, the combination of substitution mutations comprises a combination of T36A, E37R substitution mutations (M4-3, SEQ ID NO: 10). In one embodiment, the substitution mutations comprise a combination of Y30S, T36V, E37R substitution mutations (M4-9, SEQ ID NO: 11). In one embodiment, the substitution mutations comprise a combination of N7T, T8G, I9L, Y30A, T36V, E37R substitution mutations (M129, SEQ ID NO: 12). In one embodiment, the substitution mutations comprise a combination of N7T, T8S, Y30A, T36V, E37R substitution mutations (M120, SEQ ID NO: 13). In some embodiments, the combination of substitution mutations comprises, consists or consists essentially of a sequence as set forth in SEQ ID NO: 9, 10, 11, 12, 13, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or an amino acid sequence having one or more, preferably 2 or 3, conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

[0013] Further, the adrenergic receptor specific modulator or the muscarinic acetylcholine receptor specific modulator can further comprise a Maltose-binding protein domain (MBP), a protease cleavage site. The Maltose-binding protein domain sequence is set forth in SEQ ID NO: 14, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or an amino acid sequence having one or more, preferably 2 or 3, conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consists of thereof. The protease cleavage site includes, but is not limited to, a TEV enzyme cleavage site, a HRV 3C enzyme cleavage site, a SUMO enzyme cleavage site. The specific modulator is based on wild-type MT3 comprising N7T, T8G, T8S, I9L, Y30A, Y30S, T36A, E37R, H29T, H29E, H29S, Y30P, Y30S, Y30H, V31I, V31Y, T36V, E37R, Y52G, D53E, S54T, S54I, S54Y, S54R, S54L, S54T, and combinations of the above substitution mutations.

[0014] In some embodiments, the adrenergic receptors include, but are not limited to, α1A, α1B, α1D, α2A, α2B, and α2C. In some embodiments, the adrenergic receptors are selected from the group consisting of α1A, α1B, α1D, α2A, α2B, and α2C. In some embodiments, the muscarinic acetylcholine receptors include, but are not limited to, M1 AChR (muscarinic acetylcholine receptor M1), M2 AChR (muscarinic acetylcholine receptor M2), M3 AChR (muscarinic acetylcholine receptor M3), M4 AChR (muscarinic acetylcholine receptor M4), and M5 AChR (muscarinic acetylcholine receptor M5). In some embodiments, the muscarinic acetylcholine receptors are selected from the group consisting of M1 AChR, M2 AChR, M3 AChR, M4 AChR, and M5 AChR. In some embodiments, the adrenergic receptor-specific regulatory molecule or the muscarinic acetylcholine receptor-specific regulatory molecule can be a fusion protein comprising MBP, a protease cleavage site.

[0015] In another aspect, the present application provides a polynucleotide encoding the adrenergic receptor-specific regulatory molecule or the muscarinic acetylcholine receptor-specific regulatory molecule as described above. In another aspect, the present application provides an expression vector comprising the polynucleotide as described above. In another aspect, the present application provides a host cell comprising the expression vector as described above, which is a host cell for expressing a foreign protein, such as bacteria, yeast, insect cells, and mammalian cells. In another aspect, the present application provides a method for preparing the adrenergic receptor-specific regulatory molecule or the muscarinic acetylcholine receptor-specific regulatory molecule as described above, which comprises culturing the host cell as described above, and recovering the specific regulatory molecule from the cell culture. In another aspect, the present application provides a pharmaceutical composition comprising the adrenergic receptor-specific regulatory molecule or the muscarinic acetylcholine receptor-specific regulatory molecule as described above and a pharmaceutically acceptable carrier. Further, wherein the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0016] In another aspect, the present application provides use of the adrenergic receptor-specific regulatory molecule or the muscarinic acetylcholine receptor-specific regulatory molecule as described above in the preparation of a kit or a medicament for preventing, treating, and / or diagnosing a disorder associated with adrenergic receptors and muscarinic acetylcholine receptors. Further, the disorder associated with adrenergic receptors and muscarinic acetylcholine receptors is selected from central nervous system-related disorders, such as hypertension, arrhythmia, Alzheimer's disease, Parkinson's disease, etc.

[0017] Further specifically, the present application provides the following: the present application provides an adrenaline receptor specificity regulating molecule specifically binding to adrenaline receptor alpha 2A. The complex structure of muscarine toxin 3 (MT3) and adrenaline receptor alpha 2A is analyzed by cryo-EM method, according to the analyzed complex cryo-EM structure model, it is determined that H29, Y30, V31, Y35, T36, E37 on Finger 2 and Y52, D53, S54 on Finger 3 of MT3 play an important role in the binding with the receptor. According to the complex model, the inventors introduce directional mutations in the Finger 2 region and the Finger 3 region of MT3, and construct a yeast display library. Seven specific targeting adrenaline receptor alpha 2A proteins are screened and obtained in the present application, and the proteins are biotinylated, and the biotinylated receptor proteins are used for in vitro screening. Seven specific regulating molecules are isolated in the present application, and are named as A64, A66, RD7, A2S5, A2-3, A2-6, A2-10. Further, the present application determines that the seven specific regulating molecules have specificity in the adrenaline receptor family and the muscarine acetylcholine receptor family by flow cytometry method. The specificity refers to that the specific regulating molecules only bind to adrenaline receptor alpha 2A, and do not bind to other receptors of the same subtype.

[0018] The present application provides a muscarine acetylcholine receptor specificity regulating molecule specifically binding to muscarine acetylcholine receptor M4. First, the complex structure of muscarine toxin 3 (MT3) and muscarine acetylcholine receptor M4 is analyzed by cryo-EM method, according to the analyzed complex cryo-EM structure model, it is determined that N7, T8, I9 on Finger 1 and H29, Y30, V31, Y35, T36, E37 on Finger 2 of MT3 play an important role in the binding with the receptor. According to the complex model, the inventors introduce directional mutations in the Finger 1 region and the Finger 2 region of MT3, and construct a yeast display library. Five specific targeting muscarine acetylcholine receptor M4 proteins are isolated and obtained in the present application, and the proteins are biotinylated, and the biotinylated receptor proteins are used for in vitro screening. Five specific regulating molecules are isolated in the present application, and are named as M4S3, M4-3, M4-9, M129, M120. Further, the present application determines that the five specific regulating molecules have specificity in the adrenaline receptor family and the muscarine acetylcholine receptor family by flow cytometry method. The specificity refers to that the specific regulating molecules only bind to human muscarine acetylcholine receptor M4, and do not bind to other receptors of the same subtype.

[0019] The beneficial effects of this invention are as follows: Firstly, the specific regulatory molecules in this invention can all bind to the target receptor with high affinity. Flow cytometry experiments show that A2S5, A2-3, A2-6, and RD7 among the specific regulatory molecules of adrenaline receptor α2A bind to the EC5 of adrenaline receptor α2A. 50 The values ​​were 13.9 nM, 74.2 nM, 28.2 nM, and 73.2 nM, respectively; the EC50 values ​​of M4S3, M4-3, M4-9, M120, and M129, which are specific regulatory molecules of the muscarinic acetylcholine receptor M4, are related to the binding of M4S3, M4-3, M4-9, M120, and M129 to the muscarinic acetylcholine receptor M4. 50 The values ​​were 15.9 nM, 107.2 nM, 49.6 nM, 11.7 nM, and 11.2 nM, respectively. Furthermore, the specific regulatory molecules of this invention can bind to the orthotopic sites of adrenaline receptor α2A and muscarinic acetylcholine receptor M4, competing with receptor agonists for binding sites and inhibiting receptor activation, thereby effectively inhibiting receptor-mediated G protein signaling pathways. G protein dissociation experiments showed that A2S5, A2-3, and A2-6 can effectively inhibit agonist-mediated activation of adrenaline receptor α2A, with IC50 values ​​of 15.9 nM, 107.2 nM, 49.6 nM, 11.7 nM, and 11.2 nM. 50 The effective concentrations were 4.8 nM, 30.6 nM, and 11.9 nM, respectively; M4S3, M4-3, and M4-9 can effectively inhibit agonist-mediated activation of muscarinic acetylcholine receptor M4, with IC50 values ​​of 4.8 nM, 30.6 nM, and 11.9 nM. 50 The values ​​are 15.4 nM, 52.7 nM, and 85.2 nM, respectively. Attached Figure Description

[0020] Figure 1 A schematic diagram of the MT3 mutation site in a yeast display library targeting the adrenaline receptor α2A. Figure 1 A) and a diagram of the library construction ( Figure 1 A) and a schematic diagram of the screening process for adrenaline receptor α2A-specific regulatory molecules ( Figure 1 B).

[0021] Figure 2 A schematic diagram of the MT3 mutation site in a yeast display library targeting the muscarinic acetylcholine receptor M4. Figure 2 A) and a diagram of the library construction ( Figure 2 A) and a schematic diagram of the screening process for muscarinic acetylcholine receptor M4-specific regulatory molecules ( Figure 2 B).

[0022] Figure 3SDS-PAGE gel electrophoresis results of adrenergic receptor α2A specific regulatory molecule fusion protein in the application are shown; wherein lane Marker is marker, lanes 1~14 are MBP-3C-A64 sample, biotinylated MBP-3C-A64 sample after incubation with excess Streptavidin, MBP-3C-A66 sample, biotinylated MBP-3C-A66 sample after incubation with excess Streptavidin, MBP-3C-RD7 sample, biotinylated MBP-3C-RD7 sample after incubation with excess Streptavidin, MBP-3C-A2S5 sample, biotinylated MBP-3C-A2S5 sample after incubation with excess Streptavidin, MBP-3C-A2-3 sample, biotinylated MBP-3C-A2-3 sample after incubation with excess Streptavidin, MBP-3C-A2-6 sample, biotinylated MBP-3C-A2-6 sample after incubation with excess Streptavidin, MBP-3C-A2-10 sample, biotinylated MBP-3C-A2-10 sample after incubation with excess Streptavidin in turn.

[0023] Figure 4 SDS-PAGE gel electrophoresis results of muscarinic acetylcholine receptor M4 specific regulatory molecule fusion protein in the application are shown; wherein lane Marker is marker, lanes 1~12 are MBP-3C-MT3 sample, biotinylated MBP-3C-MT3 sample after incubation with excess Streptavidin, MBP-3C-M4S3 sample, biotinylated MBP-3C-M4S3 sample after incubation with excess Streptavidin, MBP-3C-M4-3 sample, biotinylated MBP-3C-M4-3 sample after incubation with excess Streptavidin, MBP-3C-M4-9 sample, biotinylated MBP-3C-M4-9 sample after incubation with excess Streptavidin, MBP-3C-M129 sample, biotinylated MBP-3C-M129 sample after incubation with excess Streptavidin, MBP-3C-M120 sample, biotinylated MBP-3C-M120 sample after incubation with excess Streptavidin in turn.

[0024] Figure 5The binding ability of adrenaline receptor α2A specific regulatory molecules on the yeast level to adrenaline receptor α2A and muscarinic acetylcholine receptor M4 is shown; wherein Figure A shows the binding ability of RD7, A64, A66, MT3 and A91 as controls; Figure B shows the binding ability of A2-3, A2-6, A2-10, MT3 as control; Figure C shows the binding ability of A2S5.

[0025] Figure 6 The specificity, affinity and biochemical function characterization results of adrenaline receptor α2A specific regulatory molecule A2S5 are shown; wherein Figure A shows the binding ability of A2S5 to adrenaline receptor subtypes and muscarinic acetylcholine receptor subtypes; Figure B shows the affinity curve of A2S5 and MT3, A2S5 is purple and MT3 is black; the curve is fitted with a "1:1 binding" model; Figure C shows the G protein-coupled receptor dissociation kinetics curve of A2S5 and MT3, A2S5 is purple and MT3 is black, the curve is fitted with a "four parameters" model; Figure D shows the blocking of MT3, A2S5 and M4S3 to adrenaline receptor α2A mediated β-arrestin signaling pathway under three concentration gradients.

[0026] Figure 7 The affinity of adrenaline receptor α2A specific regulatory molecules A2-3, A2-6, RD7 and the biochemical function characterization results of A2-3 and A2-6 are shown; wherein Figure A shows the affinity curve of A2-3 and A2-6, the curve is fitted with a "1:1 binding" model; Figure B shows the affinity curve of RD7, the curve is fitted with a "1:1 binding" model; Figure C shows the G protein-coupled receptor dissociation kinetics curve of A2-3 and A2-6, the curve is fitted with a "four parameters" model.

[0027] Figure 8 The binding ability of muscarinic acetylcholine receptor M4 specific regulatory molecules on the yeast level to adrenaline receptor α2A and muscarinic acetylcholine receptor M4 is shown; wherein Figure A shows the binding ability of M4S3, M129, M120, MT3 as control, Figure B shows the binding ability of M4-3, M4-9.

[0028] Figure 9The specificity, affinity and biochemical function characterization results of muscarinic acetylcholine receptor M4 specific regulation molecule M4S3 are shown; wherein Figure A shows the binding ability of M4S3 in adrenaline receptor subtypes and muscarinic acetylcholine receptor subtypes; Figure B shows the affinity curves of M4S3 and MT3, M4S3 is red, MT3 is black, and the curve is fitted with a "1:1 binding" model; Figure C shows the G protein coupled receptor dissociation kinetics curves of M4S3 and MT3, M4S3 is red, MT3 is black, and the curve is fitted with a "four parameters" model; Figure D shows the blocking of MT3, A2S5 and M4S3 on the muscarinic acetylcholine receptor M4 mediated β-arrestin signal pathway under three concentration gradients.

[0029] Figure 10 The affinity and biochemical function characterization results of muscarinic acetylcholine receptor M4 specific regulation molecules M4-3 and M4-9 are shown; wherein A shows the affinity curves of M4-3 and M4-9, and the curve is fitted with a "1:1 binding" model; Figures B and C show the G protein coupled receptor dissociation kinetics curves of M4-3 and M4-9, and the curve is fitted with a "four parameters" model.

[0030] Figure 11 The affinity curves of muscarinic acetylcholine receptor M4 specific regulation molecules M129 and M120 are shown, and the curve is fitted with a "1:1 binding" model. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0032] Example 1. Construction of a yeast display library targeting adrenaline receptor α2A

[0033] The present application uses yeast surface display technology to screen and obtain specific regulatory molecules that can specifically recognize adrenaline receptor alpha2A. The method is based on the characteristics that known toxin protein MT3 has been proved to exhibit high affinity to a variety of G protein-coupled receptors including adrenaline receptor alpha2A and muscarinic acetylcholine receptor M4, and its unique "three-finger folding" structure has the plasticity characteristics in structure. The specific implementation is as follows: first, the complex structure of toxin protein MT3 and adrenaline receptor alpha2A is analyzed by cryo-EM method, and according to the analyzed complex cryo-EM structure model, it is determined that the sites H29, Y30, V31, Y35, T36, E37 on Finger 2 and the sites Y52, D53, S54 on Finger 3 of MT3 play an important role in the binding with the receptor. According to the complex model, the inventors introduce directional mutations in the Finger 2 region and the Finger 3 region of MT3, and construct a yeast display library.

[0034] The yeast library is screened by using magnetic bead sorting (MACS) and flow fluorescence cell sorting (FACS) method, and the detailed screening process is as follows Figure 1The screening process is shown in Figure B, which includes four rounds of iterative screening, and the specific implementation is as follows: in the first round, 1 μM biotinylated muscarinic acetylcholine receptor M4 and adrenergic receptor a1A protein are incubated with the yeast library, and streptavidin magnetic beads (Miltenyi Biotec, 130-048-101) are added for negative selection. The magnetic beads capture and remove non-specific binding clones, and the unbound yeast cells are collected and incubated with 1 μM biotinylated adrenergic receptor a2A protein and streptavidin magnetic beads. After the target binding clones are captured by the magnetic beads, they are eluted and expanded; in the second round, the first round of expanded library is used, and biotinylated muscarinic acetylcholine receptor M4 and adrenergic receptor a1A protein are used for magnetic bead negative selection, and the process is the same as the first round of screening. Then, 1 μM biotinylated adrenergic receptor a2A protein is added to the unbound cells, and FITC-labeled anti-c-Myc antibody (Miltenyi Biotec, 130-116-485) is added to detect the expression of the displayed protein, and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) is added to detect the binding of a2A protein. The double-positive population is collected by flow cytometry (Beckman, CytoFlexSRT) and expanded; in the third round, the second round of expanded library is incubated with 0.5 μM non-biotinylated muscarinic acetylcholine receptor M4 and adrenergic receptor a1A protein, and cross-reactive clones are removed by competitive binding. Add 0.1 μM biotinylated adrenergic receptor a2A protein and the above FITC-labeled anti-c-Myc antibody and Alexa Fluor 647-labeled streptavidin, and flow sort the double-positive population; in the fourth round, the third round of expanded library is incubated with 0.5 μM non-biotinylated muscarinic acetylcholine receptor M4 and adrenergic receptor a1A protein, and cross-reactive clones are removed by competitive binding. Add 0.05 μM biotinylated adrenergic receptor a2A protein and the above FITC-labeled anti-c-Myc antibody and Alexa Fluor 647-labeled streptavidin, and flow sort the double-positive population to obtain a high-affinity clone population. Finally, the high-affinity clone population is subjected to single clone sequencing, and finally a specific regulatory molecule that specifically binds to adrenergic receptor a2A protein is obtained. Seven target adrenergic receptor a2A specific regulatory molecules are obtained by screening, and are named A64, A66, RD7, A2S5, A2-3, A2-6, A2-10, respectively, and their sequences are SEQ ID NO: 2, 3, 4, 5, 6, 7, 8.

[0035] The selection of mutation sites and the construction of yeast display library are as follows Figure 1As shown in A, the library construction is achieved by the following steps: first, design a mutant primer, the forward primer targets the MT3 domain (SEQ ID NO: 28), and introduces a degenerate codon NNK at positions 29, 30, 31, 35, 36, 37; the reverse primer targets the MT3 domain (SEQ ID NO: 29), and synchronously introduces NNK degenerate codons at positions 52, 53, 54; second, obtain the MT3 gene fragment containing the combination mutation by PCR amplification using Taq DNA polymerase (self-made); third, linearize the modified pYD1 vector (Addgene, #73447) by double enzyme digestion with BamH1 and Xba1 (ThermoFisher, K1991); fourth, transform the fragment and the linearized vector into the EBY100 yeast strain (ATCC MYA-4941) by the Gene Pulser Xcell system (Bio-Rad), and obtain the yeast display library targeting adrenergic receptor α2A by amplification and induction.

[0036] Example 2. Construction of a yeast display library targeting muscarinic acetylcholine receptor M4

[0037] The present application uses yeast surface display technology screening to obtain specific regulatory molecules that can specifically recognize muscarinic acetylcholine receptor M4. This method is based on the characteristics that the known toxin protein MT3 has been shown to exhibit high affinity for a variety of G protein-coupled receptors including adrenergic receptor α2A and muscarinic acetylcholine receptor M4, and its unique "three-finger fold" structure has the characteristic of structural plasticity. The specific implementation is as follows: first, the complex structure of toxin protein MT3 and muscarinic acetylcholine receptor M4 is analyzed by cryo-EM method, and according to the analyzed complex cryo-EM structure model, it is determined that the sites N7, T8, I9 on Finger 1 and H29, Y30, V31, Y35, T36, E37 on Finger 2 of MT3 play an important role in binding with the receptor. According to the complex model, the inventors introduced directional mutations in the Finger 1 region and the Finger 2 region of MT3, and constructed a yeast display library.

[0038] The yeast library is subjected to multiple rounds of screening by magnetic bead sorting (MACS) and flow fluorescence cell sorting (FACS), and the detailed flow chart is as follows Figure 2As shown in B, the screening process is iterated for five rounds, and the specific embodiments are as follows: in the first round, 1 μM biotinylated adrenergic receptor α1A and adrenergic receptor α2A proteins are incubated with the yeast library, streptavidin magnetic beads (Miltenyi Biotec, 130-048-101) are added for negative selection, the magnetic beads capture and remove non-specific binding clones, the unbound yeast cells are collected, 1 μM biotinylated muscarinic acetylcholine receptor M4 protein and streptavidin magnetic beads are added for incubation, the target binding clones are captured by the magnetic beads, and then eluted and amplified; in the second round, the first round of amplified library is used, biotinylated adrenergic receptor α1A and adrenergic receptor α2A proteins are used for magnetic bead negative selection, and the screening method is the same as the first round. Then, 0.5 μM biotinylated muscarinic acetylcholine receptor M4 protein is added to the unbound cells, and FITC-labeled anti-c-Myc antibody (Miltenyi Biotec, 130-116-485) is added to detect the expression of the displayed protein, Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) is added to detect the binding of the muscarinic acetylcholine receptor M4 protein, and the double-positive population is collected by a flow cytometry (Beckman, CytoFlex SRT) and amplified; in the third round, the second round of amplified library is incubated with 0.5 μM non-biotinylated adrenergic receptor α1A and adrenergic receptor α2A proteins, cross-reactive clones are removed by competitive binding, 0.2 μM biotinylated muscarinic acetylcholine receptor M4 protein, and the above-mentioned FITC-labeled anti-c-Myc antibody and Alexa Fluor 647-labeled streptavidin are added, and the double-positive population is sorted by flow cytometry; in the fourth round, the third round of amplified library is incubated with 0.5 μM non-biotinylated adrenergic receptor α1A and adrenergic receptor α2A proteins, cross-reactive clones are removed by competitive binding, 0.1 μM biotinylated muscarinic acetylcholine receptor M4 protein, and the above-mentioned FITC-labeled anti-c-Myc antibody and Alexa Fluor 647-labeled streptavidin are added, and the double-positive population is sorted by flow cytometry to obtain a high-affinity clone population; in the fifth round, the fourth round of amplified library is incubated with 1 μM non-biotinylated adrenergic receptor α1A and adrenergic receptor α2A proteins, cross-reactive clones are removed by competitive binding, 0.05 μM biotinylated muscarinic acetylcholine receptor M4 protein and the above FITC-labeled anti-c-Myc antibody and Alexa Fluor 647-labeled streptavidin, flow-sorted double-positive population to obtain a high-affinity clone group; finally, the high-affinity clone group was subjected to monoclonal sequencing, and finally a specific regulatory molecule specifically binding to the muscarinic acetylcholine receptor M4 protein was obtained, and five specific regulatory molecules targeting the muscarinic acetylcholine receptor M4 protein were screened, and were named M4S3, M4-3, M4-9, M129, and M120, respectively, and their sequences were SEQ ID NO: 9, 10, 11, 12, and 13, respectively.

[0039] The selection of the mutation site and the construction of the yeast display library are as shown in Figure 2 A, and the library construction is achieved by the following steps: first, design a mutation primer, the forward primer targets the MT3 domain (SEQ ID NO: 48) and introduces a degenerate codon NNK at positions 7, 8, and 9; the reverse primer targets the MT3 domain (SEQ ID NO: 49) and simultaneously introduces NNK degenerate codons at positions 29, 30, 31, 35, 36, and 37; second, use Taq DNA polymerase (self-made) to amplify the MT3 gene fragment containing the combination mutation by PCR; third, linearize the modified pYD1 vector (Addgene, #73447) by double enzyme digestion with BamH1 and Xba1 (ThermoFisher, K1991); fourth, transform the fragment and the linearized vector into the EBY100 yeast strain (ATCC MYA-4941) by the Gene Pulser Xcell system (Bio-Rad), and obtain the yeast display library targeting the muscarinic acetylcholine receptor M4 by amplification and induction.

[0040] Example 3. Expression, purification, and biotinylation of the obtained specific regulatory molecules

[0041] (1) Design general primers (SEQ ID NO: 30~33) for constructing fusion expression vectors. Specifically, the sequence of the a2A-specific regulatory molecule (amino acid sequence SEQ ID NO: 2~8, nucleotide sequence SEQ ID NO: 34~40) and the sequence of the M4-specific regulatory molecule (amino acid sequence SEQ ID NO: 9~13, nucleotide sequence SEQ ID NO: 41~45) are fused with the sequence of the MBP protein (amino acid sequence SEQ ID NO: 14, nucleotide sequence SEQ ID NO: 50). Introduce a 3C protease recognition cleavage site (amino acid sequence SEQ ID NO: 16, nucleotide sequence SEQ ID NO: 51) between MBP and the specific regulatory molecule, and add a secretion signal peptide (SEQ ID NO: 15, nucleotide sequence SEQ ID NO: 52) and an 8xHis purification tag at the N-terminus for purification. After amplifying the above-mentioned fragments by primers, clone them into the pcDNA3.1(+) plasmid (Invitrogen, V79020) to obtain recombinant plasmids for expressing each fusion protein.

[0042] (2) Transfect the above-mentioned plasmids into mammalian cells Expi293F™ Cells (Thermo Fisher, Cat#A14527) using PEI (Polysciences, Inc. Cat#24765) by transient transfection, and collect the culture supernatant after 5 days of culture. Purify the antibody in the supernatant using an NI-NTA resin (Yeason, 20502ES50) column. The purification steps are as follows: collect the cell culture supernatant expressing the MBP-a2A-specific regulatory molecule fusion protein or the MBP-M4-specific regulatory molecule fusion protein, and add 1 / 10 volume of 10x PBS buffer to it. Incubate the mixture with the NI-NTA resin (Yeason, 20502ES50) at 4°C for 2 hours. Then, elute and collect the target protein using 1x PBS buffer containing 300 mM imidazole. Concentrate the obtained target protein and perform SDS-PAGE gel electrophoresis, and the results are shown in Figure 3 and 4 After expression and purification, high-purity MBP-a2A-specific regulatory molecule fusion protein or MBP-M4-specific regulatory molecule fusion protein is obtained from the supernatant, respectively.

[0043] (3) Biotinylation of the fusion protein. Specifically, the fusion protein was diluted to 5 mg / mL and incubated with EZ-link™ NHS-Biotin (Thermo Fisher, CAS: 20217) at 4°C overnight, and then the fusion protein was passed through a molecular sieve to remove excess biotin. The labeling efficiency was evaluated by SDS-PAGE gel electrophoresis, and the protein labeling efficiency was as shown in FIGS. 1A and 1B. Figure 3 , Figure 4

[0044] Example 4. Preliminary characterization of specificity of adrenergic receptor a2A specific regulatory molecule

[0045] The DNA sequence (SEQ ID NO: 34~40) expressing the adrenergic receptor a2A specific regulatory molecule was constructed into the plasmid pYD1 (Addgene, #73447) of the yeast surface display system, transformed into the EBY100 yeast strain (ATCC MYA-4941) by chemical transformation, and after induction, displayed on the surface of yeast. The biotinylated adrenergic receptor a2A protein (SEQ ID NO: 20) and the muscarinic acetylcholine receptor M4 protein (SEQ ID NO: 26) were used to incubate with the yeast to detect whether they were bound. Specifically, 10 7 μL of yeast screening solution was added to resuspend the cells. Each protein sample was incubated with 50 μL of yeast cells at 4°C for 2 h, and after incubation, the cells were washed to remove the unbound protein, and the washing was repeated three times. Then, an appropriate volume of FITC-labeled anti-c-Myc antibody (Miltenyi Biotec, 130-116-485) and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) was added, and the mixture was incubated at 4°C for 1 h. After the incubation, the yeast cells were washed to remove the excess fluorescent antibody, and then flow cytometry analysis was performed (FIG. 2), the FITC fluorescence channel was used to detect the expression of the specific regulatory molecule displayed on the surface of the yeast, and the APC fluorescence channel was used to detect the binding signal of the specific regulatory molecule and the target sample protein. It was found that the adrenergic receptor a2A specific regulatory molecules A64, A66, RD7, A2S5, A2-3, A2-6 and A2-10 only bound to the biotinylated adrenergic receptor a2A protein, but not to the biotinylated muscarinic acetylcholine receptor M4 protein. Figure 5

[0046] Example 5. Characterization of the affinity and binding epitope of the adrenergic receptor a2A specific regulatory molecule using A2S5 as an example

[0047] ​​(1) The specificity binding ability of adrenergic receptor a2A specific regulation molecule A2S5 is preliminarily characterized by flow cytometry combined with experiments. The specificity refers to that the specific regulation molecules only bind to adrenergic receptor a2A, and do not bind to other adrenergic receptor subtypes and muscarinic acetylcholine receptor subtypes. First, the wild type plasmids of each subtype of adrenergic receptor (SEQ ID NO: 17~22) and each subtype of muscarinic acetylcholine receptor (SEQ ID NO: 23~27) are transfected into HEK293T cells (ATCC CRL-3216), and the transfection process is as follows: one day in advance, the HEK293T cells are passaged into a 12-well cell culture plate, and when the cells grow to a density of 75%-90%, the preheated fresh complete culture medium (Gibco, 11965092) is used for replacement; 30-60 min after replacement, the transfection mixture is prepared; 1 μg of the target receptor plasmid (SEQ ID NO: 17~22) is diluted into 50 μL of serum-free and antibiotic-free culture medium, and after gentle blowing and uniformity, 2 μL of Lipo8000 transfection reagent (Bi Yun Tian, C0533) is added, and after mixing again, it is incubated at room temperature for 5 min; the mixed solution after incubation is evenly dropped into the 12-well plate cells, and they are placed in a 37 ℃ incubator for culture. After the cells express the target receptor, flow cytometry binding experiment is carried out, and the specific operation is as follows: after transfection for 24-48 h, the cells are treated with trypsin digestion, and centrifuged at 500 g for 5 min; the cell pellet is resuspended with PBS, and the cells are washed repeatedly for 3 times; biotin-labeled specific regulation molecule A2S5 is added to the cell sample, and after the addition of the protein, it is incubated on ice for 2 h; centrifugation is performed to remove the supernatant, and PBS solution is added to wash the cells to remove excess toxin protein; fluorescent antibody APC-labeled anti-Flag antibody (Miltenyi Biotec, 130-135-514) and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) are added to the cell sample, respectively, and incubated on ice for 1 h; the cells are centrifuged and washed, and flow cytometry analysis experiment is carried out. The fluorescence signal of APC-labeled anti-Flag antibody represents the expression amount of transfected GPCR, and the fluorescence signal value of Alexa Fluor 647-labeled streptavidin represents the binding of specific regulation molecules to GPCR, and the experimental results are shown in FIG. A2S5 only binds to adrenergic receptor a2A. Figure 6 A as shown. It is detected that A2S5 only binds to adrenergic receptor a2A.

[0048] (2) Adopting flow cytometry to characterize the affinity of the adrenergic receptor a2A specific regulatory molecule. First, the wild type plasmid of adrenergic receptor a2A is transfected into HEK293T cells, and the transfection process is as follows: one day in advance, the cells are passaged into 12-well cell culture plates, and when the cells grow to 75%-90% density, the preheated fresh complete culture medium is used for replacement; 30-60 min after replacement, the transfection mixture is prepared; 1 μg of adrenergic receptor a2A plasmid is diluted into 50 μL of serum-free and antibiotic-free culture medium, and after gentle blowing and uniformity, 2 μL of Lipo 8000 transfection reagent is added, and after mixing again, it is incubated at room temperature for 5 min; the incubated mixture is evenly dropped into the 12-well plate cells, and it is placed in a 37 ℃ incubator for culture. After the cells express the target receptor, flow cytometry binding experiment is carried out, and the specific operation is as follows: after transfection for 24-48 h, the cells are treated with trypsin digestion, and centrifuged at 500 g for 5 min; the cell pellet is resuspended with PBS, and the cells are washed repeatedly for 3 times; biotin-labeled A2S5 and control protein MT3 are added to the cell sample, and each specific regulatory molecule and MT3 is gradient diluted for 10 times, and after the protein is added, it is incubated on ice for 2 h; centrifugation is performed to remove the supernatant, and PBS solution is added to wash the cells to remove excess toxin protein; fluorescent antibody anti-flag-APC (Miltenyi Biotec, 130-135-514) and SAV-A647 (Invitrogen, S21374) are added to the cell sample respectively, and incubated on ice for 1 h; centrifugation is performed to wash the cells, and flow cytometry analysis experiment is carried out, the fluorescence signal of anti-flag-APC represents the expression amount of transfected GPCR, and the fluorescence signal value of SAV-A647 represents the binding of specific regulatory molecule to GPCR, and after processing the binding value, nonlinear fitting is carried out to obtain the EC 50 of each specific regulatory molecule. The experimental results are shown in Figure 6 B. The determination results show that the EC 50 values of specific regulatory molecule A2S5 and MT3 are 13.9 nM and 18.5 nM respectively, indicating that they have comparable ligand binding affinity.

[0049] Example 6. Characterization of the antagonistic effect of adrenergic receptor a2A specific regulatory molecule A2S5 on adrenergic receptor a2A mediated signal pathway

[0050] (1) The NanoBiT experiment was used to characterize the antagonistic effect of the adrenergic receptor α2A-specific regulatory molecule A2S5 on the G protein signaling pathway. NanoBiT technology is a method for detecting intracellular protein interactions based on the principle of bioluminescence. Its core technology lies in using two structurally complementary fragments of NanoLuc luciferase (Promega), Large BiT (LgBiT) and Small BiT (SmBiT), to identify and report the interaction between two target proteins. When the target proteins interact, the fused LgBiT and SmBiT fragments approach each other and achieve structural complementarity, thereby assembling into a complete NanoLuc luciferase with bioluminescent activity. After the addition of the luciferase substrate, this complex can catalyze a luminescent reaction and release a detectable light signal. G proteins have Gα, Gβ, and Gγ subunits, and GPCRs are G protein-coupled receptors. This invention uses NanoBiT technology to monitor the dynamic dissociation process of Gα and Gβ / Gγ subunits during G protein activation in cells in real time. The specific experimental procedure is as follows: Cell transfection was performed using plasmids with a ratio of Gα:Gβ:Gγ:GPCR = 1:5:5:2; cells were collected after 24 h, cell counts were performed, and the cells were diluted to 1×10⁻⁶. 6 / mL, dispensing 80 μL / well into 96-well white plates; baseline measurement: adding 20 μL of substrate Furimazine (MCE, HY-111497) to each well and detecting the baseline value using a microplate reader; response measurement: adding different concentration gradients of the small molecule agonist brimonidine (Taoshu Biotechnology, T20157) and measuring the activation response value of GPCRs to determine the optimal agonist brimonidine response concentration; refilling the 96-well plate with cell samples, adding serially diluted A2S5 and MT3, and incubating the cells at room temperature for 2 h; adding substrate and measuring the baseline value; adding the optimal response concentration of agonist brimonidine and measuring the GPCR response value; normalizing the response values ​​and performing nonlinear fitting. In this experiment, MT3 served as the control group. The experimental results are as follows: Figure 6 As shown in C. Upon testing, the A2S5 IC... 50 The IC is 4.8 nM, MT3 50 The value was 9.3 nM. This indicates that A2S5 binds to the orthogenetic site of the receptor and acts as an antagonist in the G protein signaling pathway.

[0051] (2) Adopting the prokaryotic two-component signal transduction system (Two-Component System, TCS) to evaluate the antagonistic effect of adrenergic receptor a2A specific regulatory molecules on the adrenergic receptor a2A mediated β-arrestin recruitment. The recruitment of β-arrestin (KEGG hsa409) is an important link in the process of GPCR signal transduction. It not only participates in the desensitization and internalization of the receptor, but also plays a role in the activation of various signal pathways. The specific strategy adopted is to fuse the histidine kinase mutant NarXCA (SEQ ID NO: 47) with the C-terminal of adrenergic receptor a2A to construct a2A-N mut chimera, and fuse the mutant NarXDHP (SEQ ID NO: 46) with the C-terminal of human β-arrestin2 to construct β-arrestin2-H mut chimera. When the agonist activates the adrenergic receptor a2A, the β-arrestin2-H mut is recruited to the a2A-N mut , triggering the phosphorylation of the response regulator response protein (RR) (SEQ ID NO: 53), thereby activating the downstream reporter gene. The specific experimental procedure is as follows: the HEK293T cells are subcultured into a six-well plate one day in advance; when the cells grow to the appropriate density, the transfection system is prepared according to the ratio of a2A-N mut : β-arrestin-H mut : RR = 2:2:1, and the total plasmid amount is 2.5 micrograms per well for six-well plate cell transfection; 24 h after transfection, the cells are trypsinized and resuspended in 1.5 mL of medium, and 50 μL / well is divided into a 96-well plate, and the final concentration of 10 μM, 1 μM, 0.1 μM of A2S5, MT3, and M4S3 protein is added to each experimental group; 2 h later, the optimal stimulating concentration of the receptor agonist brimonidine (Taotuo Biological, T20157) is added, and 24 h later, the expression of the reporter gene is observed under a fluorescence microscope. In this experiment, MT3 is the positive control, M4S3 is the negative control, and the experimental results are shown in Figure 6 D. A2S5 and MT3 can inhibit the recruitment of β-arrestin, while M4S3 cannot inhibit the recruitment of β-arrestin, indicating that A2S5 can specifically inhibit the signal pathway of adrenergic receptor a2A.

[0052] Example 7. Adopting flow cytometry to characterize the affinity of adrenergic receptor a2A specific regulatory molecules RD7, A2-3, A2-6 and the antagonistic effect of A2-3, A2-6 on the G protein signal pathway.

[0053] The affinity of RD7, A2-3, A2-6 fusion proteins purified in vitro and biotinylated (prepared by the method described in Example 2) was detected using the experimental method of Example 4, and the experimental results are shown in Table 1. Figure 7 As shown in Table 1, the EC50value of RD7 was 73.2 nM, the EC50value of A2-3 was 74.2 nM, and the EC50value of A2-6 was 28.2 nM. 50 50 50

[0054] The antagonistic effect of A2-3, A2-6 fusion proteins purified in vitro (prepared by the method described in Example 2) on the G protein signaling pathway was detected using the experimental method of Example 5, and the experimental results are shown in Table 2. Figure 7 As shown in Table 2, the IC50value of A2-3 was 30.6 nM, and the IC50value of A2-6 was 11.9 nM. This indicates that A2-3 and A2-6 have an antagonistic effect on the G protein signaling pathway. 50 50

[0055] Example 8. Preliminary characterization of the specificity of muscarinic acetylcholine receptor M4-specific regulatory molecules

[0056] The DNA sequences (SEQ ID NOs: 41-45) expressing muscarinic acetylcholine receptor M4-specific regulatory molecules were constructed into the plasmid pYD1 of the yeast surface display system, transformed into EBY100 yeast cells by chemical transformation, and after induction, displayed on the surface of the yeast. The biotinylated adrenaline receptor α2A protein and muscarinic acetylcholine receptor M4 protein were used to incubate with the yeast to detect whether they were bound. The specific operation is as follows: 10 7 μL of yeast cells were incubated at 4°C for 2 h, and after incubation, the proteins not bound to the yeast cells were removed by washing three times. Then, an appropriate amount of volume of fluorescent antibody FITC-labeled anti-c-Myc antibody (Miltenyi Biotec, 130-116-485) and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) was added, and incubated at 4°C for 1 h. After the end, the yeast cells were washed to remove excess fluorescent antibody, and then flow cytometry analysis experiment was performed (BD FACS Canto II, BD Biosciences, 645876). Figure 8 ​​​​​), FITC fluorescence channel detects the expression amount of yeast surface displayed toxin protein, and APC fluorescence channel detects the binding signal of toxin protein and target sample protein. After detection, muscarinic acetylcholine receptor M4 specific regulatory molecules M4S3, M129, M120, M4-3 and M4-9 only bind to biotinylated muscarinic acetylcholine receptor M4, and do not bind to biotinylated adrenaline receptor α2A.

[0057] Example 9. Affinity and binding epitope of muscarinic acetylcholine receptor M4 specific regulatory molecule M4S3 are characterized

[0058] (1) The specific binding ability of the muscarinic acetylcholine receptor M4 specific regulatory molecule is preliminarily characterized by flow cytometry. The specificity refers to that the specific regulatory molecule only binds to the muscarinic acetylcholine receptor M4, and does not bind to other adrenaline receptor subtypes and muscarinic acetylcholine receptor subtypes. First, the wild type plasmids of adrenaline receptor subtypes (SEQ ID NO: 17-22) and muscarinic acetylcholine receptor subtypes (SEQ ID NO: 23-27) are transfected into HEK293T cells, and the transfection process is as follows: one day in advance, the HEK293T cells are passaged into a 12-well cell culture plate, and when the cells grow to a density of 75%-90%, the preheated fresh complete culture medium is used for replacement; 30-60 min after replacement, the transfection mixture is prepared; 1 μg of target GPCR plasmid is diluted into 50 μL of serum-free and antibiotic-free culture medium, and after gentle blowing and mixing, 2 μL of Lipo8000 transfection reagent is added, and after mixing again, it is incubated at room temperature for 5 min; the incubated mixture is evenly dropped into the 12-well plate cells, which are placed in a 37 ℃ incubator for culture. After the cells express the target receptor, flow cytometry binding experiment is carried out, and the specific operation is as follows: after transfection for 24-48 h, the cells are treated with trypsin, and centrifuged at 500 g for 5 min; the cell pellet is resuspended with PBS, and the cells are washed repeatedly for 3 times; biotinylated specific regulatory molecule M4S3 is added to the cell sample, and after the addition of the protein, it is incubated on ice for 2 h; the supernatant is centrifuged and washed with PBS to remove excess toxin protein; fluorescent antibody APC-labeled anti-Flag antibody (Miltenyi Biotec, 130-135-514) and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) are added to the cell sample, respectively, and incubated on ice for 1 h; the cells are centrifuged and washed, and flow cytometry analysis is carried out. The fluorescence signal of APC-labeled anti-Flag antibody represents the expression amount of transfected GPCR, and the fluorescence signal value of Alexa Fluor 647-labeled streptavidin represents the binding of specific regulatory molecule to GPCR. The experimental results are as follows: Figure 9As shown in A. It was detected that M4S3 only binds to muscarinic acetylcholine receptor M4, but not to other subtypes.

[0059] (2) The affinity of the muscarinic acetylcholine receptor M4 specific regulatory molecule was characterized by flow cytometry. First, the wild-type plasmid of the muscarinic acetylcholine receptor M4 was transfected into HEK293T cells, and the transfection process was as follows: the cells were passaged into 12-well cell culture plates one day in advance, and when the cells grew to 75%-90% density, the cells were washed with preheated fresh complete culture medium; 30-60 min after the medium was changed, the transfection mixture was prepared; 1 μg of muscarinic acetylcholine receptor M4 plasmid was diluted into 50 μL of serum-free and antibiotic-free medium, and then 2 μL of Lipo 8000 transfection reagent was added after gentle blowing and mixing, and the mixture was incubated at room temperature for 5 min; the incubated mixture was evenly dropped into the cells in the 12-well plate, and the plate was placed in a 37 ℃ incubator for culture. After the cells expressed the target receptor, flow cytometry binding experiment was performed, and the specific operation was as follows: after transfection for 24-48 h, the cells were treated with trypsin and centrifuged at 500 g for 5 min; the cell pellet was resuspended with PBS and washed repeatedly for 3 times; biotin-labeled specific regulatory molecule M4S3 and control protein MT3 were added to the cell sample, and M4S3 and MT3 were diluted in 10 gradients; after the protein was added, it was incubated on ice for 2 h; the supernatant was removed by centrifugation, and the cells were washed with PBS solution to remove excess toxin protein; fluorescent antibody APC-labeled anti-Flag antibody (Miltenyi Biotec, 130-135-514) and Alexa Fluor 647-labeled streptavidin (Invitrogen, S21374) were added to the cell sample, respectively, and incubated on ice for 1 h; the cells were washed by centrifugation, and flow cytometry analysis was performed. The fluorescence signal of APC-labeled anti-Flag antibody represents the expression amount of transfected GPCR, and the fluorescence signal value of Alexa Fluor 647-labeled streptavidin represents the binding of specific regulatory molecules to GPCR. After processing the binding values, the EC50 of each specific regulatory molecule was obtained by nonlinear fitting, and the experimental results are as follows: Figure 9 As shown in B. It was detected that the EC50 of M4S3 was 15.9 nM, and the EC50 of MT3 was 16.7 nM, indicating that the two had comparable ligand binding affinities. 50 50

[0060] Example 10. Characterization of the antagonistic effect of M4S3 on the muscarinic acetylcholine receptor M4-mediated signal pathway

[0061] ​​(1) The NanoBiT assay was used to characterize the antagonistic effect of muscarinic acetylcholine receptor M4-specific regulatory molecules on the G protein signaling pathway. The specific experimental procedure was as follows: Cells were transfected according to the plasmid ratio of Gα:Gβ:Gγ:GPCR=1:5:5:2; cells were collected after 24 h, cell counts were performed, and the cells were diluted to 1×10⁻⁶. 6 / mL, dispensing 80 μL / well into 96-well white plates; baseline measurement: adding 20 μL of substrate Furimazine to each well and detecting the baseline value using a microplate reader; response measurement: adding different concentration gradients of the small molecule agonist bethanechol chloride (Taoshu Biotechnology, T3126) and measuring the activation response value of GPCRs to determine the optimal agonist bethanechol chloride response concentration; refilling the 96-well plates with cell samples, and adding serially diluted M4S3 and MT3 respectively, and incubating the cells at room temperature for 2 h; adding substrate and measuring the baseline value; adding the optimal response concentration of agonist bethanechol chloride and measuring the GPCR response value; normalizing the response values ​​and performing nonlinear fitting. In this experiment, MT3 served as the control group. The experimental results are as follows: Figure 9 As shown in C. Upon testing, the M4S3 IC... 50 The IC of MT3 is 15.4 nM. 50 The value was 35.4 nM. This indicates that M4S3 binds to the orthogenetic site of the receptor and acts as an antagonist in the G protein signaling pathway.

[0062] (2) The antagonistic effect of muscarinic acetylcholine receptor M4-specific regulatory molecules on muscarinic acetylcholine receptor M4-mediated β-arrestin recruitment was evaluated using a prokaryotic two-component signal transduction system (TCS). The specific strategy employed was to fuse the histidine kinase mutant NarXCA (SEQ ID NO: 47) with the C-terminus of the muscarinic acetylcholine receptor M4 to construct M4-N mut A chimera was constructed by fusing the mutant NarXDHP (SEQ ID NO: 46) with the C-terminus of human β-arrestin2 to construct β-arrestin2-H. mut Chimera. The specific experimental procedure is as follows: HEK293T cells were passaged into six-well plates one day in advance; after the cells grew to a suitable density, they were cultured according to the M4-N... mut β-arrestin-H mut: RR=2:2:1 plasmid amount for six-well plate cell transfection; 24 h after transfection, trypsin digestion of cells, resuspension of cells with 1.5 mL of medium, and according to the amount of 50 μL / well, 96-well plates were divided, and the final concentration of 10 μM, 1 μM, 0.1 μM of A2S5, MT3, and M4S3 proteins were added in each experimental group; 2 h later, the optimal stimulating concentration of receptor agonist bethanechol chloride (Taozhuo Biotechnology, T3126) was added, and 24 h later, the expression of the reporter gene was observed by fluorescence microscopy. In this experiment, MT3 was the positive control, and A2S5 was the negative control. The experimental results are shown in Figure 9 D. M4S3 and MT3 can inhibit the recruitment of β-arrestin, while A2S5 cannot inhibit the recruitment of β-arrestin, indicating that M4S3 can specifically inhibit the signal pathway of muscarinic acetylcholine receptor M4.

[0063] Example 11. Characterization of muscarinic acetylcholine receptor M4 specific regulatory molecules M4-3, M4-9, M129, and M120 by flow cytometry combined with experiments and NanoBiT experiments, respectively, to characterize the affinity of M4-3, M4-9, M129, and M120 and the antagonistic effect of M4-3 and M4-9 on the G protein signal pathway

[0064] The affinity of M4-3, M4-9, M129, and M120 fusion proteins purified in vitro and biotinylated (prepared by the method described in Example 2) was detected, and the experimental method was the same as that in Example 9. The experimental results are shown in Figure 10 A and Figure 11 It was detected that the EC 50 value of M4-3 was 107.2 nM, the EC 50 value of M4-9 was 49.6 nM, the EC 50 value of M129 was 11.2 nM, and the EC 50 value of M120 was 11.7 nM.

[0065] The antagonistic effect of M4-3 and M4-9 fusion proteins purified in vitro (prepared by the method described in Example 2) on the G protein signal pathway was detected, and the experimental method was the same as that in Example 10. The experimental results are shown in Figure 10 B and Figure 10 C. It was detected that the IC 50 value of M4-3 was 52.7 nM, and the IC 50 value of M4-9 was 85.2 nM. This indicates that M4-3 and M4-9 have an antagonistic effect on the G protein signal pathway.

[0066] SEQUENCE LISTING

[0067] SEQ ID NO: 1 muscarinic toxin 3 (MT3) amino acid sequence

[0068] LTCVTKNTIFGITTENCPAGQNLCFKRWHYVIPRYTEITRGCAATCPIPENYDSIHCCKTDKCNE

[0069] SEQ ID NO: 2 A64 amino acid sequence

[0070] LTCVTKNTIFGITTENCPAGQNLCFKRWTPVIPRYVEITRGCAATCPIPENGDIIHCCKTDKCNE

[0071] SEQ ID NO: 3 A66 amino acid sequence

[0072] LTCVTKNTIFGITTENCPAGQNLCFKRWTSIIPRYVEITRGCAATCPIPENGDYIHCCKTDKCNE

[0073] SEQ ID NO: 4 RD7 amino acid sequence

[0074] LTCVTKNTIFGITTENCPAGQNLCFKRWEYYIPRYVRITRGCAATCPIPENGDYIHCCKTDKCNE

[0075] SEQ ID NO: 5 A2S5 amino acid sequence

[0076] LTCVTKNTIFGITTENCPAGQNLCFKRWSHVIPRYTEITRGCAATCPIPENGDRIHCCKTDKCNE

[0077] SEQ ID NO: 6 A2-3 amino acid sequence

[0078] LTCVTKNTIFGITTENCPAGQNLCFKRWHYVIPRYTEITRGCAATCPIPENGDRIHCCKTDKCNE

[0079] SEQ ID NO: 7 A2-6 amino acid sequence

[0080] LTCVTKNTIFGITTENCPAGQNLCFKRWHYVIPRYTEITRGCAATCPIPENGDLIHCCKTDKCNE

[0081] SEQ ID NO:8 A2-10 amino acid sequence

[0082] LTCVTKNTIFGITTENCPAGQNLCFKRWHYVIPRYTEITRGCAATCPIPENGETIHCCKTDKCNE

[0083] SEQ ID NO:9 M4S3 amino acid sequence

[0084] LTCVTKNTIFGITTENCPAGQNLCFKRWHAVIPRYVRITRGCAATCPIPENYDSIHCCKTDKCNE

[0085] SEQ ID NO:10 M4-3 amino acid sequence

[0086] LTCVTKNTIFGITTENCPAGQNLCFKRWHYVIPRYARITRGCAATCPIPENYDSIHCCKTDKCNE

[0087] SEQ ID NO:11 M4-9 amino acid sequence

[0088] LTCVTKNTIFGITTENCPAGQNLCFKRWHSVIPRYVRITRGCAATCPIPENYDSIHCCKTDKCNE

[0089] SEQ ID NO:12 M129 amino acid sequence

[0090] LTCVTKTGLFGITTENCPAGQNLCFKRWHAVIPRYVRITRGCAATCPIPENYDSIHCCKTDKCNE

[0091] SEQ ID NO:13 M120 amino acid sequence

[0092] LTCVTKTSIFGITTENCPAGQNLCFKRWHAVIPRYVRITRGCAATCPIPENYDSIHCCKTDKCNE

[0093] SEQ ID NO:14 maltose binding protein amino acid sequence

[0094] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

[0095] SEQ ID NO: 15 Secretion signal peptide amino acid sequence

[0096] MNLLLILTFVAAAVA

[0097] SEQ ID NO: 16 HRV 3C cleavage site amino acid sequence

[0098] LEVLFQGP

[0099] SEQ ID NO: 17 Epinephrine alpha 1A receptor expression sequence

[0100] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLNANFTGPNQTSSNSTLPQLDITRAISVGLVLGAFILFAIVGNILVILSVACNRHLRTPTNYFIVNLAMADLLLSFTVLPFSAALEVLGYWVLGRIFCDIWAAVDVLCCTASILSLCAISIDRYIGVRYSLQYPTLVTRRKAILALLSVWVLSTVISIGPLLGWKEPAPNDDKECGVTEEPFYALFSSLGSFYIPLAVILVMYCRVYIVAKRTTKNLEAGVMKEMSNSKELTLRIHSKNFHEDTLSSTKAKGHNPRSSIAVKLFKFSREKKAAKTLGIVVGMFILCWLPFFIALPLGSLFSTLKPPDAVFKVVFWLGYFNSCLNPIIYPCSSKEFKRAFVRILGCQCGSHHHHHHHH

[0101] SEQ ID NO: 18 Epinephrine alpha 1 B receptor expression sequence

[0102] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLNANFTGPNQTSSNSTLPQLDITRAISVGLVLGAFILFAIVGNILVILSVACNRHLRTPTNYFIVNLAMADLLLSFTVLPFSAALEVLGYWVLGRIFCDIWAAVDVLCCTASILSLCAISIDRYIGVRYSLQYPTLVTRRKAILALLSVWVLSTVISIGPLLGWKEPAPNDDKECGVTEEPFYALFSSLGSFYIPLAVILVMYCRVYIVAKRTTKNLEAGVMKEMSNSKELTLRIHSKNFHEDTLSSTKAKGHNPRSSIAVKLFKFSREKKAAKTLGIVVGMFILCWLPFFIALPLGSLFSTLKPPDAVFKVVFWLGYFNSCLNPIIYPCSSKEFKRAFVRILGCQCGSHHHHHHHH

[0103] SEQ ID NO: 19 Epinephrine alpha 1 D receptor expression sequence

[0104] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLMFRQEQPLAEGSFAPMGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLTVFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMGYWYFGKAWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAIIITVWVISAVISFPPLISIEKKGGGGGPQPAEPRCEINDQKWYVISSCIGSFFAPCLIMILVYVRIYQIAKRRTRVPPSRRGPDAVAAGPGEERVGAAKASRWRGRQNREKRFTFVLAVVIGVFVVCWFPFFFTYTLTAVGCSVPRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIVGSHHHHHHHH

[0105] SEQ ID NO: 20 Epinephrine alpha 2A receptor expression sequence

[0106] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLMFRQEQPLAEGSFAPMGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLTVFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMGYWYFGKAWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAIIITVWVISAVISFPPLISIEKKGGGGGPQPAEPRCEINDQKWYVISSCIGSFFAPCLIMILVYVRIYQIAKRRTRVPPSRRGPDAVAAGPGEERVGAAKASRWRGRQNREKRFTFVLAVVIGVFVVCWFPFFFTYTLTAVGCSVPRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIVGSHHHHHHHH

[0107] SEQ ID NO: 21 Epinephrine alpha 2B receptor expression sequence

[0108] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLMDHQDPYSVQATAAIAAAITFLILFTIFGNALVILAVLTSRSLRAPQNLFLVSLAAADILVATLIIPFSLANELLGYWYFRRTWCEVYLALDVLFCTSSIVHLCAISLDRYWAVSRALEYNSKRTPRRIKCIILTVWLIAAVISLPPLIYKGDQGPQPRGRPQCKLNQEAWYILASSIGSFFAPCLIMILVYLRIYLIAKRSNRRGPRAKGGPGQGEQWWRRRAQLTREKRFTFVLAVVIGVFVLCWFPFFFSYSLGAICPKHCKVPHGLFQFFFWIGYCNSSLNPVIYTIFNQDFRRAFRRILCRPWTQTAWGSHHHHHHHH

[0109] SEQ ID NO: 22 Epinephrine alpha 2C receptor expression sequence

[0110] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLGGVANASGASWGPPRGQYSAGAVAGLAAVVGFLIVFTVVGNVLVVIAVLTSRALRAPQNLFLVSLASADILVATLVMPFSLANELMAYWYFGQVWCGVYLALDVLFCTSSIVHLCAISLDRYWSVTQAVEYNLKRTPRRVKATIVAVWLISAVISFPPLVSLYRQPDGAAYPQCGLNDETWYILSSCIGSFFAPCLIMGLVYARIYRVAKLRTRTLSEKRAPVGPDGASPTTENFLSRRRRARSSVCRRKVAQAREKRFTFVLAVVMGVFVLCWFPFFFSYSLYGICREACQVPGPLFKFFFWIGYCNSSLNPVIYTVFNQDFRRSFKHILGSHHHHHHHH

[0111] SEQ ID NO: 23 Muscarinic acetylcholine receptor Ml expression sequence

[0112] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLMNNSTNSSNNSLALTSPYKTFEVVFIVLVAGSLSLVTIIGNILVMVSIKVNRHLQTVNNYFLFSLACADLIIGVFSMNLYTLYTVIGYWPLGPVVCDLWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPVKRTTKMAGMMIAAAWVLSFILWAPAILFWQFIVGVRTVEDGECYIQFFSNAAVTFGTAIAAFYLPVIIMTVLYWHISRASKSRIKKDKKEPVANQDPVSIVARKIVKMTKQPAKKKPPPSREKKVTRTILAILLAFIITWAPYNVMVLINTFCAPCIPNTVWTIGYWLCYINSTINPACYALCNATFKKTFKHLLMCHYKNIGATRGSHHHHHHHH

[0113] SEQ ID NO: 24 Muscarinic acetylcholine receptor M2 expression sequence

[0114] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLMNNSTNSSNNSLALTSPYKTFEVVFIVLVAGSLSLVTIIGNILVMVSIKVNRHLQTVNNYFLFSLACADLIIGVFSMNLYTLYTVIGYWPLGPVVCDLWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPVKRTTKMAGMMIAAAWVLSFILWAPAILFWQFIVGVRTVEDGECYIQFFSNAAVTFGTAIAAFYLPVIIMTVLYWHISRASKSRIKKDKKEPVANQDPVSIVARKIVKMTKQPAKKKPPPSREKKVTRTILAILLAFIITWAPYNVMVLINTFCAPCIPNTVWTIGYWLCYINSTINPACYALCNATFKKTFKHLLMCHYKNIGATRGSHHHHHHHH

[0115] SEQ ID NO: 25 Muscarinic acetylcholine receptor M3 expression sequence

[0116] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQALGSHHHHHHHH

[0117] SEQ ID NO: 26 Muscarinic acetylcholine receptor M4 expression sequence

[0118] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLSAMANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSLVTVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKGYWPLGAVVCDLWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTKMAGLMIAAAWVLSFVLWAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAAFYLPVVIMTVLYIHISLASRSRVHKHRPEGPKEKKAVARKFASIARNQVRKKRQMAARERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDTVWSIGYWLCYVNSTINPACYALCNATFKKTFRHLLLCQGSHHHHHHHH

[0119] SEQ ID NO: 27 Muscarinic acetylcholine receptor M5 expression sequence

[0120] MKTIIALSYIFCLVFADYKDDDDKGSEQKLISEEDLEGDSYHNATTVNGTPVNHQPLERHRLWEVITIAAVTAVVSLITIVGNVLVMISFKVNSQLKTVNNYYLLSLACADLIIGIFSMNLYTTYILMGRWALGSLACDLWLALDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTPKRAGIMIGLAWLISFILWAPAILCWQYLVGKRTVPLDECQIQFLSEPTITFGTAIAAFYIPVSVMTILYCRIYRETEKRTKDLADLQGSDSVTKAEKRKPAHRALFKEPSTKGLNPNPSHQMTKRKRVVLVKERKAAQTLSAILLAFIITWTPYNIMVLVSTFCDKCVPVTLWHLGYWLCYVNSTVNPICYALCNRTFRKTFKMLLLCRWKKKKVEEKLYWQGNSKLPGSHHHHHHHH

[0121] SEQ ID NO: 28 Forward mutagenic primer nucleotide sequence for a yeast display library targeting adrenergic receptor alpha 2A (5’-3’)

[0122] TGTTTTAAGAGATGGNNKNNKNNKATCCCAAGGNNKNNKNNKATCACCCGGGGGTGCGC

[0123] SEQ ID NO: 29 Reverse mutagenic primer nucleotide sequence for a yeast display library targeting adrenergic receptor alpha 2A (5’-3’)

[0124] CACTTATCTGTTTTACAACAATGTATMNNMNNMNNGTTTTCGGGGATAGGGCA

[0125] SEQ ID NO: 30 Forward primer Fl nucleotide sequence for alpha 2A specific regulatory molecule expression plasmid (5’-3’)

[0126] CGCGGCCGCAGTCGCCCATCACCATCACCACCATCACCATATGAAAATAGAAGAAGGA

[0127] SEQ ID NO: 31 Forward primer F2 nucleotide sequence for M4 specific regulatory molecule expression plasmid (5’-3’)

[0128] AAGTCCTTTTCCAAGGCCCTCTGACCTGCGTGACAAA

[0129] SEQ ID NO: 32 Reverse primer Rl nucleotide sequence (5'-3') for the α2A-specific regulatory molecule expression plasmid

[0130] GCCTTGGAAAAGGACTTCCAGAGAACCTGTCTGGGCGTCCTTCA

[0131] SEQ ID NO: 33 Reverse primer R2 nucleotide sequence (5'-3') for the M4-specific regulatory molecule expression plasmid

[0132] ATGGCGGCCAAGCTGGGGATCCTCACTCATTACACTTATCTGT

[0133] SEQ ID NO: 34 A64 nucleotide sequence

[0134] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGACCCCGGTGATCCCAAGGTACGTGGAAATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATATTATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0135] SEQ ID NO: 35 A66 nucleotide sequence

[0136] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGACCAGCATTATCCCAAGGTACGTGGAAATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATTATATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0137] SEQ ID NO: 36 RD7 nucleotide sequence

[0138] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGGAATATTATATCCCAAGGTACGTGCGCATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATTATATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0139] SEQ ID NO: 37 A2S5 nucleotide sequence

[0140] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGAGCCATGTGATCCCAAGGTACACCGAAATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATCGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0141] SEQ ID NO: 38 A2-3 nucleotide sequence

[0142] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACTATGTCATCCCAAGGTACACGGAGATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATCGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0143] SEQ ID NO: 39 A2-6 nucleotide sequence

[0144] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACTATGTCATCCCAAGGTACACGGAGATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGATAAGATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0145] SEQ ID NO: 40 A2-10 nucleotide sequence

[0146] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACTATGTCATCCCAAGGTACACGGAGATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACGGCGAAACCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0147] SEQ ID NO: 41 M4S3 nucleotide sequence

[0148] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACGCGGTCATCCCAAGGTACGTGCGCATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACTACGACAGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0149] SEQ ID NO: 42 M4-3 nucleotide sequence

[0150] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACTATGTCATCCCAAGGTACGCGCGCATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACTACGACAGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0151] SEQ ID NO:43 M4-9 nucleotide sequence

[0152] CTGACCTGCGTGACAAAAAACACAATTTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACTCGGTCATCCCAAGGTACGTGCGCATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACTACGACAGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0153] SEQ ID NO:44 M129 nucleotide sequence

[0154] CTGACCTGCGTGACAAAAACCGGCCTGTTCGGAATTACCACTGAAAACTGCCCGGCTGGCCAGAATCTCTGTTTTAAGAGATGGCACGCGGTCATCCCAAGGTACGTGCGCATCACCCGGGGGTGCGCAGCCACTTGCCCTATCCCCGAAAACTACGACAGCATACATTGTTGTAAAACAGATAAGTGTAATGAG

[0155] SEQ ID NO:45 M120 nucleotide sequence

[0156] CTTATCTCCGAAGAAGACTTGGGTAGCGGCTCTGGATCCCTGACCTGCGTGACAAAANNKNNKNNKTTCGGAATTACCACTGA

[0157] SEQ ID NO: 46 Histidine kinase mutant NarX DHP amino acid sequence

[0158] QERQQQLIVMEERATIARELQDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

[0159] SEQ ID NO: 47 Histidine kinase mutant NarX CA amino acid sequence

[0160] QERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSAALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

[0161] SEQ ID NO: 48 Forward mutation primer nucleotide sequence for yeast display library targeting muscarinic acetylcholine receptor M4 (5’-3’)

[0162] CTTATCTCCGAAGAAGACTTGGGTAGCGGCTCTGGATCCCTGACCTGCGTGACAAAANNKNNKNNKTTCGGAATTACCACTGA

[0163] SEQ ID NO: 49 Reverse mutant primer nucleotide sequence for yeast display library targeting muscarinic acetylcholine receptor M4 (5’-3’)

[0164] GCGCACCCCCGGGTGATMNNMNNMNNCCTTGGGATMNNMNNMNNCCATCTCTTAAAACA

[0165] SEQ ID NO: 50 Maltose binding protein nucleotide sequence

[0166]

[0167] SEQ ID NO: 51 HRV 3C enzyme cleavage site nucleotide sequence

[0168] CTGGAAGTCCTTTTCCAAGGCCCT

[0169] SEQ ID NO: 52 Secretion signal peptide nucleotide sequence

[0170] ATGAATCTCCTGCTAATTCTTACGTTCGTCGCGGCCGCAGTCGCC

[0171] SEQ ID NO: 53 Response regulator response protein (RR) amino acid sequence

[0172] MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF

Claims

1. An adrenergic receptor specific modulator molecule, which specifically binds to the adrenergic receptor a2A, preferably to the human adrenergic receptor a2A; wherein the adrenergic receptor specific modulator molecule comprises one or more substitution mutations based on wild-type MT3, the amino acid sequence of which is shown in SEQ ID NO: 1, selected from the group consisting of H29T, H29E, H29S, Y30P, Y30S, Y30H, V31I, V31Y, T36V, E37R, Y52G, D53E, S54T, S54I, S54Y, S54R, S54L, S54T; Preferably, the adrenergic receptor specific modulator molecule does not bind to an adrenergic receptor other than the adrenergic receptor a2A, optionally does not bind to a muscarinic acetylcholine receptor; The adrenergic receptor other than the adrenergic receptor a2A includes, but is not limited to, the adrenergic receptors a1A, a1B, a1D, a2B and a2C; The muscarinic acetylcholine receptor includes, but is not limited to, the muscarinic acetylcholine receptors M1, M2, M3, M4 and M5.

2. The adrenergic receptor specific modulator molecule of claim 1, which has the amino acid sequence shown in SEQ ID NO:

5.

3. The adrenergic receptor specific modulator molecule of claim 1, which has an amino acid sequence comprising: (1) a sequence as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8; (2) a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a sequence as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8; (3) an amino acid sequence which has one or more, preferably 2 or 3, conservative amino acid substitutions, insertions or deletions compared to a sequence as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8; or (4) consists of a sequence as described in (1) - (3).

4. A muscarinic acetylcholine receptor specific modulator molecule, which specifically binds to the muscarinic acetylcholine receptor M4, preferably to the human muscarinic acetylcholine receptor M4; wherein the muscarinic acetylcholine receptor specific modulator molecule comprises one or more substitution mutations based on wild-type MT3, the amino acid sequence of which is shown in SEQ ID NO: 1, selected from the group consisting of N7T, T8G, T8S, I9L, Y30A, Y30S, T36V, T36A, E37R; Preferably, the muscarinic acetylcholine receptor specific modulator molecule does not bind to a muscarinic acetylcholine receptor other than the muscarinic acetylcholine receptor M4, optionally does not bind to an adrenergic receptor; The muscarinic acetylcholine receptors other than the muscarinic acetylcholine receptor M4 include, but are not limited to, the muscarinic acetylcholine receptors M1, M2, M3 and M5; The adrenergic receptors include, but are not limited to, the adrenergic receptors a1A, a1B, a1D, a2A, a2B and a2C.

5. The muscarinic acetylcholine receptor specific modulator of claim 4, having an amino acid sequence as set forth in SEQ ID NO:

9.

6. The muscarinic acetylcholine receptor specific modulator of claim 4, having an amino acid sequence comprising: (a) a sequence as set forth in SEQ ID NO: 9, 10, 11, 12 or 13; (b) a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a sequence as set forth in SEQ ID NO: 9, 10, 11, 12 or 13; or (c) an amino acid sequence having one or more, preferably 2 or 3, conservative amino acid substitutions insertions or deletions compared to a sequence as set forth in SEQ ID NO: 9, 10, 11, 12 or 13; or (d) consisting of a sequence as set forth in (a)-(c).

7. A polynucleotide encoding the adrenergic receptor specific modulator of any one of claims 1-3 or the muscarinic acetylcholine receptor specific modulator of any one of claims 4-6.

8. An expression vector comprising the polynucleotide of claim 7, or expressing the adrenergic receptor specific modulator of any one of claims 1-3 or the muscarinic acetylcholine receptor specific modulator of any one of claims 4-6.

9. A host cell comprising the expression vector according to claim 8, which is a host cell for expression of a foreign protein, such as a bacterial, yeast, insect cell and mammalian cell.

10. A method for preparing an adrenergic receptor specific modulator as defined in any one of claims 1 to 3 or a muscarinic acetylcholine receptor specific modulator as defined in any one of claims 4 to 6, characterized in that, comprising culturing the host cell of claim 9, and recovering the adrenergic receptor specific modulator or the muscarinic acetylcholine receptor specific modulator from the cell culture.

11. A pharmaceutical composition or kit comprising the adrenergic receptor specific modulator of any one of claims 1-3 or the muscarinic acetylcholine receptor specific modulator of any one of claims 4-6, preferably further comprising a pharmaceutically acceptable carrier, preferably further comprising a second antibody which specifically recognizes the specific modulator, optionally the second antibody comprising a detectable label, preferably a radioisotope, a luminescent substance, a colored substance, an enzyme or a polyethylene glycol.

12. Use of the adrenergic receptor specific modulator of any one of claims 1 to 3 or the muscarinic acetylcholine receptor specific modulator of any one of claims 4 to 6 for the manufacture of a kit or medicament for the prevention, treatment and / or diagnosis of a disease or disorder associated with an adrenergic receptor and / or a muscarinic acetylcholine receptor, wherein the disease or disorder associated with an adrenergic receptor and / or a muscarinic acetylcholine receptor comprises a central nervous system related disorder, preferably hypertension, arrhythmia, Alzheimer's disease, Parkinson's disease.

13. A fusion protein comprising the adrenergic receptor specific modulator of any one of claims 1 to 3 or the muscarinic acetylcholine receptor specific modulator of any one of claims 4 to 6, the fusion protein further comprising a maltose binding protein domain MBP and / or a protease cleavage site; the sequence of the maltose binding protein domain MBP is: (A) the sequence as shown in SEQ ID NO: 14, (B) a sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 14, (C) an amino acid sequence which has one or more, preferably 2 or 3, conservative amino acid substitutions, insertions or deletions compared to the sequence shown in SEQ ID NO: 14, or (D) consists of a sequence as described in (A) to (C); the protease cleavage site comprises, but is not limited to, a TEV enzyme cleavage site, a HRV 3C enzyme cleavage site or a SUMO enzyme cleavage site. ​ ​ ​ ​ ​

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