A halo-tag-based oriented covalent immobilization cell membrane chromatographic column, its preparation method and application

Abstract

The application discloses a Halo-tag-based directional covalent immobilization cell membrane chromatographic column and a preparation method and application thereof, and belongs to the technical field of chromatography science.The Halo-tag-based directional covalent immobilization cell membrane chromatographic column is prepared by the following steps: bonding haloalkane to silica gel, then utilizing high specificity recognition and stable covalent combination between the Halo-tag and the substrate haloalkane, fixing the cell membrane on the surface of the silica gel stationary phase, obtaining a cell membrane chromatographic stationary phase, and finally wetly packing the cell membrane chromatographic stationary phase to obtain the Halo-tag-based directional covalent immobilization cell membrane chromatographic column.The Halo-tag-based directional covalent immobilization cell membrane chromatographic column prolongs the column life of the cell membrane chromatographic column, obviously improves the stability of the cell membrane chromatographic column within one week, and to some extent, solves the problem of poor specificity of the ordinary cell membrane chromatographic column, thereby providing technical support for the wide application of the cell membrane chromatographic column.

Description

Technical Field

[0001] This invention belongs to the field of affinity chromatography science and technology, specifically relating to a Halo-tag-based directional covalently immobilized cell membrane chromatography column and its preparation method and application. Background Technology

[0002] Cell membrane chromatography involves immobilizing cell membranes onto a carrier to create a stationary phase. Chromatographic techniques are then used to study the interactions between drugs and receptors in the mobile phase. It is also a technique that integrates chromatographic separation and activity recognition, and it has unique advantages in screening active ingredients in complex systems.

[0003] Traditional cell membrane chromatography mainly employs two methods of cell membrane immobilization: physical adsorption and chemical bonding. The drawbacks of traditional cell membrane chromatography columns prepared using silica gel adsorption are: because the cell membrane and silica gel are primarily bound by weak forces such as hydrophobic interactions and van der Waals forces, the cell membrane gradually detaches from the silica gel surface during use, leading to reduced column efficiency and a shorter lifespan. Furthermore, the complex types of cell surface receptors make it difficult to identify the specific receptors of potential active drugs, and the specificity of cell membrane chromatography columns still needs further improvement. Chemical bonding cell membrane immobilization, combined with the use of genetically engineered cells that highly express specific receptors, mitigates some of these shortcomings to some extent. However, commonly used non-specific chemical bonds and methods often affect the activity of some receptors, even causing membrane receptors to lose their recognition activity, thus limiting its widespread application.

[0004] Conventional non-directional chemical fixation methods may reduce or even eliminate the activity of the target protein, and also cause disorder in the fixation of the target receptor on the cell membrane. Currently, there are no reports on technologies that use Halo-tag for directional covalent fixation of cell membrane chromatography columns to solve the above problems. Therefore, there is a need for a method that does not affect the activity of the target protein, while improving the lifespan, stability and specificity of cell membrane chromatography columns. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a Halo-tag-based directional covalently immobilized cell membrane chromatography column, its preparation method and application, which improves the lifespan, stability and specificity of the cell membrane chromatography column without affecting the activity of the target protein.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention discloses a Halo-tag-based directional covalently immobilized cell membrane chromatography column, wherein the Halo-tag-based directional covalently immobilized cell membrane chromatography column is filled with a Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase;

[0008] The Halo-tag-directed covalently immobilized cell membrane chromatography stationary phase was prepared by mixing haloalkane-modified silica gel with a cell membrane solution that highly expressed Halo-tag and fused to the C-terminus / N-terminus of the target receptor protein.

[0009] This invention also discloses a method for preparing a Halo-tag-based directional covalently immobilized cell membrane chromatography column. The method involves mixing haloalkane-modified silica gel with a cell membrane solution containing a target receptor protein fused to the C-terminus / N-terminus with a high expression of Halo-tag to obtain a Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase. This Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase is then packed into a chromatography column to obtain a Halo-tag-based directional covalently immobilized cell membrane chromatography column.

[0010] Preferably, the specific steps are as follows:

[0011] 1) Haloalkanes are mixed with amino silica gel to prepare haloalkanes-modified silica gel;

[0012] 2) Culture cells with high expression of Halo-tag fused to the C-terminus / N-terminus of the target receptor protein, break up the cell pellet, centrifuge, collect the supernatant, and precipitate the cell membrane pellet. Mix the cell membrane pellet with physiological saline to obtain a cell membrane suspension. Then mix the cell membrane suspension with silica gel modified with haloalkane to prepare a directional covalently fixed cell membrane chromatographic stationary phase of Halo-tag.

[0013] 3) The Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase was packed into a column to prepare a Halo-tag-based directional covalently immobilized cell membrane chromatography column.

[0014] More preferably, in step 1), the solvent used for mixing is DMF.

[0015] Preferably, in step 1), after mixing, the mixture is filtered, washed with ultrapure water until the pH of the filtrate is neutral, and the filter cake is dried at 37°C for 12 hours to obtain silica gel modified with haloalkane.

[0016] Preferably, in step 2), the cell preparation method is as follows: construct a plasmid containing a Halo-tag fused to the C-terminus of the target receptor protein, and transfect it into cells after being loaded with lentivirus. Use the resistance gene in the plasmid to screen the transfected cells, and then use 0.25% trypsin to digest and collect cells in the logarithmic growth phase. After centrifugation at 1000g for 10 min at 4°C, discard the culture medium to obtain the cells.

[0017] Preferably, in step 2), the cell pellet is broken up by centrifugation as follows: the cell pellet is added to Tris-HCl and suspended on ice, broken up with a cell disruptor, centrifuged at 1000g for 10 min at 4°C, transferred and centrifuged at 12000g for 20 min at 4°C to obtain cell membrane pellet.

[0018] Preferably, in step 2), the cell membrane suspension is mixed with silica gel modified with haloalkane, shaken at 37°C for 1 hour, and then left to stand overnight to obtain the Halo-tag directional covalently fixed cell membrane chromatographic stationary phase.

[0019] Preferably, in step 3), the column packing is done using a wet packing method.

[0020] This invention also discloses the application of the above-mentioned Halo-tag-based directional covalently immobilized cell membrane chromatography column in studying ligand-receptor interactions and screening for potential active components acting on specific receptors.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a Halo-tag-based directional covalently immobilized cell membrane chromatography column. Halogenated alkanes are covalently bonded to silica gel, and then reacted with cell membranes fused with the C-terminus / N-terminus of a target receptor protein that highly expresses Halo-tag. The Halo-tag specifically reacts with the halogenated alkanes to form covalent bonds, thereby directionally and covalently immobilizing the target receptor on the modified silica gel surface. Compared to the low lifespan and specificity of traditional physical adsorption immobilization of cell membranes, the cell membrane chromatography column of this invention, due to the covalent bonding method used for cell membrane immobilization, can extend the time the cell membrane is on the silica gel, as well as the lifespan and stability of the cell membrane chromatography column, without affecting the activity of the target protein. By utilizing the expression position of the protein tag (C-terminus / N-terminus) and the specific reaction of Halo-tag with halogenated alkanes, directional immobilization and further screening of the cell membrane are achieved. This ensures that only receptor membrane fragments fused with Halo-tag are immobilized, further improving the specificity of the chromatography column and providing technical support for the wider application of cell membrane chromatography columns.

[0023] This invention provides a method for preparing a Halo-tag-based directional covalently immobilized cell membrane chromatography column. The preparation conditions are mild and the operation is simple, which can lay a good foundation for large-scale industrial production of cell membrane chromatography columns. Compared with traditional physically adsorbed cell membrane chromatography columns, the Halo-tag cell membrane chromatography column prepared by this method has significantly improved column stability and service life. Attached Figure Description

[0024] Figure 1This is a flowchart illustrating the preparation of the Halo-tag-based directional covalently immobilized cell membrane chromatography column of the present invention;

[0025] Figure 2 X-ray photoelectron spectra of amino silica gel (SiO2-NH2), chloroalkane-modified silica gel (SiO2-Cl), and Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase (CMSP) of the present invention;

[0026] Figure 3 Field emission scanning electron microscope (FESEM) images of aminosilicone, chloroalkane-modified silica gel, and Halo-tag-based directional covalently immobilized cell membrane chromatographic stationary phases of the present invention; wherein, a is 2 μm, aminopropyl silica gel, b is 2 μm, chloroalkane-modified aminopropyl silica gel, c is 2 μm, Halo-tag-MRGPRX2 cell membrane stationary phase, d is 1 μm, aminopropyl silica gel, e is 1 μm, chloroalkane-modified aminopropyl silica gel, and f is 1 μm, Halo-tag-MRGPRX2 cell membrane stationary phase;

[0027] Figure 4 Transmission electron microscopy (TEM) images of the chloroalkane-modified silica gel and the Halo-tag-based directional covalently immobilized cell membrane chromatographic stationary phase of the present invention; wherein, a is 200 nm, chloroalkane-modified aminopropyl silica gel, b is 200 nm, Halo-tag-MRGPRX2 cell membrane stationary phase, c is 50 nm, chloroalkane-modified aminopropyl silica gel, and d is 50 nm, Halo-tag-MRGPRX2 cell membrane stationary phase.

[0028] Figure 5 The images show the chromatograms of the sinomenine of the present invention on day 7 on a Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography system (Halo-tag-MRGPRX2 / CMC) and on day 4 on a conventional physical adsorption MRGPRX2 cell membrane chromatography system (MRGPRX2 / CMC). Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "comprising" and "having" and any variations thereof in this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0031] The present invention will now be described in further detail with reference to the accompanying drawings:

[0032] The present invention provides a Halo-tag-based directional covalently immobilized cell membrane chromatography column, wherein the Halo-tag-based directional covalently immobilized cell membrane chromatography column is filled with a Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase;

[0033] The Halo-tag-directed covalently immobilized cell membrane chromatography stationary phase was prepared by mixing haloalkane-modified silica gel with a cell membrane solution that highly expressed Halo-tag and fused to the C-terminus / N-terminus of the target receptor protein.

[0034] Compared to traditional physical adsorption cell membrane chromatography columns, this cell membrane chromatography column extends the time the cell membrane spends on silica gel without affecting the activity of the target protein, thus improving both column lifespan and stability. Simultaneously, the specific reaction of Halo-tag with haloalkanes further screens the immobilized cell membrane; only receptor membrane fragments fused to express Halo-tag are immobilized. This addresses, to some extent, the poor specificity of traditional columns. Furthermore, C-terminal / N-terminal fusion expression of Halo-tag enables directional immobilization of the cell membrane. This directional covalently immobilized Halo-tag cell membrane chromatography column provides technical support for the further widespread application of cell membrane chromatography columns.

[0035] This invention provides a method for preparing a Halo-tag-based directional covalently immobilized cell membrane chromatography column, such as... Figure 1 As shown, firstly, haloalkanes are mixed with amino silica gel to modify the silica gel, thus obtaining haloalkane-modified silica gel. Then, cells with high expression of Halo-tag fused to the C-terminus / N-terminus of the target receptor protein are cultured to prepare a cell membrane suspension. Next, the cell membrane suspension is mixed with haloalkane-modified silica gel. Halo-tag can specifically react with haloalkanes to form covalent bonds, thereby directionally and covalently immobilizing the target receptor on the silica gel surface, thus obtaining a Halo-tag-based directionally and covalently immobilized cell membrane chromatography stationary phase. Finally, the Halo-tag-based directionally and covalently immobilized cell membrane chromatography stationary phase is wet-packed onto a column to obtain a Halo-tag-based directionally and covalently immobilized cell membrane chromatography column.

[0036] The target protein is MRGPRX2, and the haloalkane is chloroalkane. The cell construct is HEK293 cells (X2-Halo-tag cells) with Halo-tag fused to the C-terminus of MRGPRX2. The specific operation steps are as follows:

[0037] 1) Preparation of chlorinated alkane-modified silica gel

[0038] Weigh 1.6 g of HATU into a 100 mL round-bottom flask as a condensing agent, add 40 mL of DMF to dissolve it, then add 400 μL of hexachlorohexanoic acid and 700 μL of the catalyst DIPEA, stir, and add 5 g of aminosilicone (model: Innoval, 5 μm). Add 10 mL of DMF (3.5% carbon loading), stir magnetically at room temperature for 4 h, filter, wash with ultrapure water until the pH of the filtrate is neutral, collect the filter cake in a watch glass and dry at 37 °C for 12 h to obtain chloroalkane-modified silica gel (SiO2-Cl).

[0039] 2) Preparation of Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase

[0040] A plasmid containing a Halo-tag fused to the C-terminus of MRGPRX2 was constructed using genetic engineering techniques. This plasmid was then transfected into HEK293 cells via lentiviral encapsulation. Cells were screened using an antibiotic resistance gene within the plasmid to obtain stably transfected HEK293 cells exhibiting high Halo-tag-MRGPRX2 expression (cells were constructed by Guangzhou Cyagen Biosciences Co., Ltd.). Cells in the logarithmic growth phase (at least 2 × 10⁻⁶ cells) were collected by digestion with 0.25% trypsin. 7 After centrifugation at 1000g for 10 min at 4℃, the culture medium was discarded to obtain cells. The cells were washed twice with physiological saline by centrifugation. The cell pellet was resuspended in 20 mmol / L Tris-HCl and placed on ice. The cells were then disrupted using a cell disruptor (power: 400W, 6 times). After centrifugation at 1000g for 10 min at 4℃, the supernatant cell membrane solution was transferred to a new centrifuge tube and centrifuged at 12000g for 20 min at 4℃ to obtain the cell membrane pellet. The cell membrane was resuspended in physiological saline and disrupted on ice using a cell disruptor (power: 400W, 6 times). The pellet was then added to 40 mg of chloroalkane-modified silica gel, shaken at 37℃ for 1 h, and allowed to stand overnight to obtain the Halo-tag directional covalently fixed cell membrane chromatography stationary phase (CMSP).

[0041] 3) Characterization of Halo-tag's directional covalently immobilized cell membrane chromatography stationary phase

[0042] X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (SEM) were used to characterize the directional covalently immobilized cell membrane chromatographic stationary phases of amino silica gel, chloroalkane-modified silica gel, and Halo-tag. Figure 2 XPS spectra of a Halo-tag-modified, covalently immobilized cell membrane chromatography stationary phase (CMSP) consisting of amino silica gel (SiO2-NH2), chloroalkane-modified silica gel (SiO2-Cl), and a silica gel substrate. In the SiO2-Cl XPS spectrum, a characteristic signal of chlorine (Cl2p) was observed at 200.6 eV. This is due to the condensation reaction between the amino group of the amino silica gel and the carboxyl group of hexachlorohexanoic acid, resulting in the bonding of chloroalkane to the silica gel. This indicates that the chloroalkane was successfully bonded to the amino silica gel support. In the CMSP XPS spectrum, the characteristic signal of chlorine (Cl2p) was significantly reduced, while a characteristic signal of phosphorus (P2p) was detected at 133.5 eV, indicating that the cell membrane was successfully immobilized on the support surface.

[0043] To further verify the above results, the surface morphology of the three materials was characterized using field emission scanning electron microscopy, and the results are as follows: Figure 3 After modification with chloroalkane, the surface of the aminosilicone stationary phase did not change significantly because the modification of small molecules has little effect on the surface morphology. However, after reacting with the cell membrane solution, the surface of the stationary phase underwent significant changes. Since the aminosilicone used is a porous silica, the surface pores were significantly masked after bonding with the cell membrane, making the surface of the stationary phase appear smooth. This is consistent with the results of transmission electron microscopy characterization of the aminosilicone stationary phase modified with chloroalkane and the Halo-tag-MRGPRX2 cell membrane stationary phase. Figure 4 The presence of a membrane on the surface of the Halo-tag-MRGPRX2 cell membrane stationary phase is more clearly visible, which also indicates the successful preparation of the Halo-tag directional covalently fixed cell membrane chromatographic stationary phase.

[0044] 5) Establishment of Halo-tag directional covalently immobilized cell membrane chromatography column and cell membrane chromatography system

[0045] The obtained Halo-tag-based directional covalently immobilized cell membrane chromatography stationary phase was wet-packed into a 10 mm × 2.0 mm (ID) column core using a column packer, and the matching column sleeve was placed in and the column sleeve nut was tightened to obtain the cell membrane chromatography stationary phase. This stationary phase was then connected to a liquid chromatography system to establish a cell membrane chromatography system based on Halo-tag-based directional covalent immobilization.

[0046] 6) Validation of the effect of Halo-tag-based directional covalently immobilized cell membrane chromatography column

[0047] The intra-column and inter-column differences between conventional MRGPRX2 cell membrane chromatography columns and Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography columns were investigated and compared using the retention time of sinomenine hydrochloride, a positive drug for the MRGPRX2 receptor, as an indicator.

[0048] After fully equilibrating both cell membrane chromatography columns, 2 μL of 0.5 mg / mL positive drug reference solution was injected six times consecutively into each column. The mobile phase was 2.5 mmol / L disodium hydrogen phosphate buffer (pH = 7.4), and the flow rate was 0.2 mL / min. The retention times of the positive drug were recorded, and the relative standard deviations (RSDs) of the six retention times on the two columns were compared. Three ordinary MRGPRX2 cell membrane chromatography columns and three Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography columns were prepared using the same method. After fully equilibrating each column, 2 μL of 0.5 mg / mL positive drug reference solution was injected into each column, and the retention times of the positive drug were recorded. The results are shown in Table 1.

[0049] Table 1. Repeatability and activity assessment of conventional cell membrane chromatography columns and Halo-tag-based directional covalently immobilized cell membrane chromatography columns.

[0050]

[0051] Table 1 shows that both the conventional MRGPRX2 cell membrane chromatography column and the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography column exhibit good intra-column and inter-column variability, making them suitable for screening and analyzing active ingredients in complex systems. Furthermore, the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography column showed a lower RSD value for positive drugs in the inter-column variability assessment, indicating better reproducibility than the conventional MRGPRX2 cell membrane chromatography column. Next, the activity time of the two cell membrane chromatography columns was investigated. After 3 and 7 days, 2 μL of 0.5 mg / mL positive drug reference solution was re-injected into both the conventional MRGPRX2 cell membrane chromatography column and the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography column, and the retention time was recorded. Three days later, sinomenine hydrochloride was still significantly retained on both the conventional MRGPRX2 cell membrane column and the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane column. However, the retention time RSD value was smaller on the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane column within three days. Seven days later, sinomenine hydrochloride was still well retained on the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane column. After three days, the peak shape on the conventional MRGPRX2 cell membrane column became distorted, showing double peaks. Figure 5This indicates that the Halo-tag-based directional covalently immobilized MRGPRX2 cell membrane chromatography column has a significantly longer lifespan and significantly improved stability.

[0052] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A Halo-tag based directed covalent immobilization of cell membrane chromatographic column, characterized in that, The Halo-tag-based oriented covalently immobilized cell membrane chromatographic column is filled with a Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase; The Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase is prepared by mixing halogenated alkane modified silica gel and a cell membrane solution of a target receptor protein C-terminal / N-terminal fusion Halo-tag high expression cell.

2. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column, characterized in that, The Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase is prepared by mixing halogenated alkane modified silica gel and a cell membrane solution of a target receptor protein C-terminal / N-terminal fusion Halo-tag high expression cell.

3. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 2, wherein, The specific steps are as follows: 1) mixing halogenated alkane and amino silica gel to prepare halogenated alkane modified silica gel; 2) culturing a target receptor protein C-terminal / N-terminal fusion Halo-tag high expression cell, crushing the cell precipitate, centrifuging, taking the supernatant, precipitating a cell membrane precipitate, mixing the cell membrane precipitate with physiological saline to prepare a cell membrane suspension, and mixing the cell membrane suspension with the halogenated alkane modified silica gel to prepare a Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase; 3) packing the Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase to prepare a Halo-tag-based oriented covalently immobilized cell membrane chromatographic column.

4. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 1), the solvent used for mixing is DMF.

5. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 1), after mixing, the mixture is filtered, washed with ultrapure water until the filtrate has a neutral pH, and then the filter cake is dried at 37°C for 12 hours to prepare halogenated alkane modified silica gel.

6. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 2), the cell preparation method is as follows: a plasmid containing a target receptor protein C-terminal fusion Halo-tag is constructed, and then the plasmid is packaged by a lentivirus and transfected into cells. The transfected cells are selected using the resistance gene in the plasmid, and the cells in the logarithmic growth phase are collected by 0.25% trypsin digestion. The culture medium is discarded after centrifugation at 4°C and 1000g for 10 minutes, and the cells are obtained.

7. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 2), the cell precipitate is crushed and centrifuged as follows: the cell precipitate is mixed with Tris-HCl and suspended on ice, crushed with a cell crusher, centrifuged at 4°C and 1000g for 10 minutes, and then centrifuged at 4°C and 12000g for 20 minutes after transfer, and the cell membrane precipitate is obtained.

8. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 2), after mixing the cell membrane suspension with the halogenated alkane modified silica gel, the mixture is oscillated at 37°C for 1 hour and then left standing overnight to obtain the Halo-tag oriented covalently immobilized cell membrane chromatographic stationary phase.

9. A method for preparing a Halo-tag based directed covalent immobilization of cell membrane chromatographic column as claimed in claim 3 wherein, In step 3), the packing is wet packing.

10. Use of the Halo-tag-based oriented covalently immobilized cell membrane chromatographic column of claim 1 in studying ligand-receptor interactions and screening potential active components that act on specific receptors.

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