CN116153412B - CHRNA7 ligand screening and application - Google Patents

Abstract

The application discloses a method for screening nicotinic cholinergic receptor alpha 7 (CHRNA 7) ligand by using raw letter and application of atractylone and 3 beta-acetoxyatractylone as the ligand of the CHRNA 7.

Description

CHRNA7 ligand screening and application

Technical Field

The application belongs to the field of bioinformatics and biotechnology, and particularly relates to a method for obtaining CHRNA7 ligand through letter generation screening, and application of atractylone/3 beta-acetoxyatractylone serving as ligand of nicotinic cholinergic receptor A7.

Background

Nicotinic cholinergic receptor a7 (CHRNA 7) is a member of the ligand-gated ion channel superfamily, mediating rapid signaling at synapses. CHRNA7 is a heteropentamer composed of homologous subunits. The proposed structure for each subunit is a conserved N-terminal extracellular domain followed by three conserved transmembrane domains, a variable cytoplasmic loop, a fourth conserved transmembrane domain and a short C-terminal extracellular domain. The protein encoded by this gene forms a homooligomeric channel, has significant permeability to calcium ions, and is the major component of the brain nicotinic receptor, which is blocked by α -bungarotoxin and is highly sensitive. Once this receptor binds acetylcholine, it undergoes extensive conformational changes, affecting all of the subunits and causing the ion-conducting channel across the plasma membrane to open. The gene is located in a region identified as the major susceptibility gene locus for juvenile myoclonus seizures and at a chromosomal location involved in the genetic transmission of schizophrenia. The most recently occurring evolutionarily partially repeated events of this region result in the inclusion of a hybridizing sequence from this gene and a new FAM7A gene. Alternative splicing results in multiple transcriptional variants. Thus, novel ligands for CHRNA7 were found to be useful in the treatment of related diseases.

Rhizoma atractylodis is a traditional Chinese medicine which is commonly used in traditional Chinese medicine, and has a long application history in China. Modern compound researches show that rhizoma atractylodis has complex chemical components and contains various compounds with biological activity. However, at present, the medium for treating diseases is still a large number of composite components, so that the corresponding pharmacological action mechanism and the structure-activity relationship thereof cannot be studied deeply. The traditional Chinese medicine has proved to have excellent therapeutic value through a great deal of clinical practice. The traditional Chinese medicines are various in variety, have a large number of excellent compounds, contain a large number of medicinal value compounds, and in order to better establish research on close combination of chemical components and pharmacological activity of rhizoma atractylodis, we combine a machine learning technology and a virtual screening technology which are developed at high speed in recent years to screen effective medicinal compounds in rhizoma atractylodis compound components.

In the prior art, the content of atractylone or 3 beta-acetoxyatractylone as CHRNA7 ligand has not been reported, and the application is proposed in view of the fact.

Summary of The Invention

The application aims at screening and finding out the compound which has medicinal value on CHRNA7 related diseases in rhizoma atractylodis, and through experiments such as molecular dynamics simulation, machine learning, feature screening, molecular docking and the like, the application finds out that atractylone or 3 beta-acetoxyatractylone (actetoxytracton) can be used as a ligand of nicotinic choline receptor A7 (CHRNA 7) for the first time, and further proposes a new application of atractylone or 3 beta-acetoxyatractylone in medicine.

Specifically, the technical scheme provided by the application is as follows:

in one aspect, the present application first provides a method of screening for a CHRNA7 ligand by letter, the method comprising the steps of:

1) Modeling the test ligand of CHRNA7 in the Pubchem database by using a machine learning model XGBoost,

2) Screening the compound contained in the rhizoma atractylodis in the TCMSP database by using the established classification model, molecular similarity and molecular docking,

3) The mode and the situation of the combination of the compound and the CHRNA7 are simulated by utilizing molecular dynamics simulation, and the atractylone or the 3 beta-acetoxyatractylone is screened and determined to be the ligand of the CHRNA 7.

Preferably, the method further comprises:

4) Verification was performed based on SPR affinity experiments.

In one aspect, the present application also provides any one of the following uses of atractylone/3β -acetoxyatractylone or its corresponding pharmaceutically acceptable salt, solvate or polymorph:

1) As a CHRNA7 ligand;

2) Use in modulating CHRNA 7;

3) The application in preparing a preparation for regulating and controlling CHRNA 7;

4) Use in the treatment or prevention of a CHRNA 7-related disease;

5) Use in the manufacture of a medicament for the treatment or prophylaxis of a CHRNA 7-related disorder.

Further, the modulation is inhibition or reduction of CHRNA7 protein expression, or activity, or function.

In one aspect, the present application also provides a method of modulating CHRNA7, comprising the step of administering atractylone/3β -acetoxyatractylone, or a corresponding pharmaceutically acceptable salt, solvate or polymorph thereof, to a sample.

Further, the sample is an in vivo or in vitro sample.

Further, the modulation is inhibition or reduction of CHRNA7 protein expression/activity/function.

Preferably, the inhibition or reduction is achieved by binding atractylone or 3β -acetoxyatractylone to the CHRNA7 protein.

In one aspect, the present application also provides a method of screening/identifying candidate inhibitors based on ligand receptor relationships, comprising any of the following steps:

analyzing the effect of the compound on the binding of atractylone or 3 beta-acetoxyatractylone to the receptor CHRNA7 to obtain a candidate inhibitor;

or screening a compound which inhibits the interaction of atractylone or 3 beta-acetoxyatractylone with CHRNA7 to obtain a candidate inhibitor.

In one aspect, the present application also provides an inhibitor capable of modulating the interaction of atractylone or 3β -acetoxyatractylone with the receptor CHRNA 7;

preferably, the inhibitor is a small molecule inhibitor, a polypeptide inhibitor, an antibody inhibitor or the like.

In one aspect, the present application also provides a pharmaceutical composition comprising a ligand for chra 7 and a pharmaceutically-acceptable carrier or excipient;

further, the ligand of the CHRNA7 is atractylone/3 beta-acetoxyatractylone or corresponding pharmaceutically acceptable salts, solvates or polymorphs thereof, etc.

Further, the corresponding pharmaceutically acceptable salts refer to salts of the compounds of the present application which are substantially non-toxic to living organisms, for example, salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, or salts with bases such as sodium hydroxide, potassium hydroxide, and the like.

Further, the term solvate refers to a combination of a compound of the present application formed by solvation with a solvent molecule, such as a combination of a compound with water to form a hydrate.

Further, the polymorphs refer to compounds of the present application that exist in different lattice forms.

Further, the carrier or excipient is not limited, such as: starch, sodium chloride, microcrystalline cellulose, sorbic acid, mannitol, and the like. The administration mode of the composition can be intravenous injection, oral administration, intramuscular injection, subcutaneous injection, skin surface injection, local injection and the like, and the dosage form of the composition can be injection, freeze-dried powder injection, injection microsphere, liposome, tablet, bilayer/multilayer tablet, buccal tablet, sublingual tablet, capsule, water aqua, powder, paste, spray, granule, soft capsule, dripping pill, gel, patch, paste and the like, wherein the injection, the freeze-dried powder injection, the tablet and the capsule are preferred.

The beneficial technical effect of this application:

the application firstly proposes atractylone or 3 beta-acetoxyatractylone as a ligand of a nicotinic cholinergic receptor A7 (CHRNA 7), researches show that the CHRNA7 receptor is related to various diseases, and therefore, medicines prepared by the ligand of the CHRNA7 can be applied to the diseases related to the CHRNA 7.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

In fig. 1, the model is evaluated through the confusion matrix, and the prediction of the test set sample has higher accuracy.

Fig. 2, evaluation of the model by ROC curve, AUC value of 0.94, demonstrates higher reliability of model prediction.

FIG. 3 shows that the accuracy of the algorithm is concentrated by 0.85 to about 0.9 through ten-fold cross validation, and the model can be used for stably and accurately carrying out classification prediction.

In fig. 4, through ten-fold cross validation, the ROC curves of the model are calculated, the AUC values are all greater than 0.85, and the average value is 0.92, which indicates that the reliability of the model is higher.

FIG. 5, comparison of the similarity of rhizoma Atractylodis compounds to the CHRNA7 ligand Nicotine; wherein, fig. 5A-5G are molecular similarities of the atractylis ovata compounds MOL000191, MOL000188, MOL000179, MOL000173, MOL000164, MOL000085 and MOL000043 (TCMSP database MOL ID), respectively.

FIG. 6, comparison of the affinity of the rhizoma Atractylodis compound to the CHRNA7 ligand BTX-A; wherein, FIGS. 6A-6G are molecular similarities of rhizoma Atractylodis compounds MOL000191, MOL000188, MOL000179, MOL000173, MOL000164, MOL000085 and MOL000043 (TCMSP database MOL ID), respectively.

FIG. 7 shows results of screening for a compound of rhizoma Atractylodis against CHRNA7 molecule, wherein A is atractylenolide IB atractylone and C is 3β -acetoxyactylone.

FIG. 8, results of molecular dynamics simulation of atractyl compound with CHRNA7 (RMSD, RMSF, gyrate), wherein A is RMSD, representing the stability of the system during the simulation; b is RMSF, which characterizes the flexibility and the movement intensity of the protein amino acid in the whole simulation process, and the flexible structure of the protein amino acid and the relation between the flexible structure of the protein and the ligand binding active amino acid can be explored by means of the RMSF; c is the Gyrate, which characterizes the compactness of the protein structure, and also can characterize the variation of the degree of looseness of the peptide chain of the protein in the simulation process by means of the radius of gyration, and the smaller the value, the more stable the protein structure.

FIG. 9, results of molecular dynamics simulation of a rhizoma Atractylodis compound and CHRNA7 (lowest energy conformation and chemical bond), wherein A is the stable conformation of MOL000043 and CHRNA7 complex at the lowest point of free energy, B is the stable conformation of MOL000164 and CHRNA7 complex at the lowest point of free energy, and C is the stable conformation of MOL000188 and CHRNA7 complex at the lowest point of free energy;

FIG. 10, results of affinity assays for nicotinic cholinergic receptor alpha 7 (CHRNa 7) protein with atractylone, wherein the top curves represent steady state fitted curves for ligand at 50,25,12.5,6.25,3.125 and 0. Mu.M concentrations, respectively.

Detailed Description

While this application may be embodied in many different forms, there are disclosed herein specific exemplary embodiments thereof which are indicative of the principles of the present application. It should be emphasized that this application is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

The following terms or definitions are provided solely to aid in the understanding of the present application. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the present application are intended to be the same as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

The terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only those embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

The term "and/or" is considered a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and/or" as used in phrases herein, such as "a and/or B", is intended to include a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The terms "for example" and "i.e." are used by way of example only, are not intended to be limiting, and should not be construed to refer to only those items explicitly recited in the specification.

The terms "or more", "at least", "exceeding", etc., such as "at least one" should be understood to include, but not be limited to, values of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 600, 700, 900, or 5000. But also any larger numbers or scores therebetween.

Conversely, the term "no more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. But also any smaller numbers or scores therebetween.

The terms "plurality," "at least two," "two or more," "at least a second," and the like should be understood to include, but are not limited to, at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 600, 700, 900, or more, 5000, or more. But also any larger numbers or scores therebetween.

The terms "about", "substantially" and "approximately" refer to a range of accuracy that one of skill in the art would understand yet still ensure that the technical effect of the feature in question is exhibited. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range and to include fractions thereof (e.g., tenths and hundredths of integers) as appropriate.

Various aspects of the disclosure are described in further detail.

1. Atractylone or 3 beta-acetoxyatractylone as CHRNA7 ligand and related application thereof

Protein CHRNA7 is a member of the ligand-gated ion channel superfamily, and can mediate rapid signaling at synapses. CHRNA7 is a heteropentamer composed of homologous subunits. The proposed structure for each subunit is a conserved N-terminal extracellular domain followed by three conserved transmembrane domains, a variable cytoplasmic loop, a fourth conserved transmembrane domain and a short C-terminal extracellular domain. The protein encoded by this gene forms a homooligomeric channel, has significant permeability to calcium ions, and is the major component of the brain nicotinic receptor, which is blocked by α -bungarotoxin and is highly sensitive. Once this receptor binds acetylcholine, it undergoes extensive conformational changes, affecting all of the subunits and causing the ion-conducting channel across the plasma membrane to open. The gene is located in a region identified as the major susceptibility gene locus for juvenile myoclonus seizures and at a chromosomal location involved in the genetic transmission of schizophrenia. The most recently occurring evolutionarily partially repeated events of this region result in the inclusion of a hybridizing sequence from this gene and a new FAM7A gene. Alternative splicing results in multiple transcriptional variants. Thus, novel ligands for CHRNA7 were found to be useful in the treatment of related diseases.

In some embodiments, the chra 7 receptor-related disorder comprises chronic kidney disease, nicotine dependency, schizophrenia, cognitive dysfunction, neurodegenerative disease, and the like.

The molecular formula of atractylone is as follows:

the molecular formula of the 3 beta-acetoxyatractylone is as follows:

however, the prior art of the relationship between atractylone/3 beta-acetoxyatractylone and CHRNA7 protein is not disclosed, and the application is verified by molecular dynamics simulation, machine learning, feature screening, molecular docking and other biological experiments and combined experiments, so that the atractylone/3 beta-acetoxyatractylone (actetoxytracton) can be used as a ligand of nicotinic cholinergic receptor A7 (CHRNA 7) for the first time.

It will be appreciated that based on the discovery of atractylone/3β -acetoxyatractylone as a ligand for CHRNA7, the art is aware of a range of pharmaceutical related uses of atractylone/3β -acetoxyatractylone or its corresponding pharmaceutically acceptable salts, solvates or polymorphs.

In some embodiments, the application comprises:

1) Use in modulating CHRNA 7;

2) The application in preparing a preparation for regulating and controlling CHRNA 7;

the term "modulate" generally encompasses the meaning of up-regulating or down-regulating in two different directions, in some cases it is understood that inhibition or enhancement, in some cases it is understood that decrease or increase, etc., and the specific interpretation is not limiting, as understood and interpreted according to the actual application context. Illustratively, in some embodiments, "modulating the CHRNA7" may be understood as inhibiting or enhancing the expression or activity of the CHRNA7, further aspects of the present application are inhibiting or reducing the expression or activity of the CHRNA7 protein.

In some embodiments, the application comprises:

3) Use in the treatment or prevention of a CHRNA 7-related disease;

4) Use in the manufacture of a medicament for the treatment or prophylaxis of a CHRNA 7-related disorder.

The term "treatment" refers to any type of intervention or procedure performed on a subject, or administration of an active agent to the subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions, or biochemical indicators associated with a disease. In some embodiments, "treating" includes partial remission. In another embodiment, "treating" includes complete remission.

It is understood that the prior art has disclosed CHRNA7 related diseases such as the above mentioned chronic kidney disease, nicotine dependency, schizophrenia, cognitive dysfunction and neurodegenerative diseases, etc. While atractylone/3 beta-acetoxyatractylone is known to be able to modulate CHRNA7, it is understood that atractylone/3 beta-acetoxyatractylone is known to be useful in therapeutic or pharmaceutical applications for these diseases. Thus the present application also includes a method of treating a CHRNA 7-related disorder by administering atractylone/3β -acetoxyatractylone or a corresponding pharmaceutically acceptable salt, solvate or polymorph thereof.

In some embodiments, the application may further comprise:

5) Use in screening and/or identifying candidate inhibitors of CHRNA 7-related diseases.

Based on the receptor ligand relationship, one skilled in the art can evaluate candidate inhibitors by analyzing the effect of compounds on the binding of 3β -acetoxyatractylone to the receptor CHRNA7 during drug or inhibitor screening; or evaluating the candidate inhibitor by screening for a compound that inhibits the interaction of 3β -acetoxyatractylone and CHRNA 7. The present application may thus also include a method of screening candidate inhibitors of the corresponding CHRNA 7-related disease by evaluating the candidate inhibitors by analyzing the effect of a compound on the binding of 3β -acetoxyatractylone to the receptor CHRNA 7; or evaluating the candidate inhibitor by screening for a compound that inhibits the interaction of 3β -acetoxyatractylone and CHRNA 7.

2. Pharmaceutical combinations and administration of the present application

A pharmaceutical composition comprising a ligand for chra 7; the ligand of the CHRNA7 is atractylone/3 beta-acetoxyatractylone or corresponding pharmaceutically acceptable salt, solvate or polymorph. The therapeutic or prophylactic effect of the disease is achieved by administering an effective dose of the pharmaceutical composition to the individual or subject.

In some embodiments, the pharmaceutical compositions or pharmaceutical formulations of the present application comprise suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, and the like, as known in the art.

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents and the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use herein can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. See also "Handbook of Pharmaceutical Excipients", fifth edition, R.C.Rowe, P.J.Seskey and s.c. owen, pharmaceutical Press, london, chicago for the use of excipients and their use. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and/or salt thereof is chemically and/or physically compatible with the other ingredients in the formulation, and physiologically compatible with the recipient.

The term "effective dose", "effective amount" or "therapeutically effective dose" is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from onset of a disease or promotes regression of a disease as evidenced by a decrease in severity of symptoms of the disease, an increase in the frequency and duration of the disease asymptomatic phase, or prevention of injury or disability due to affliction of the disease. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

As used herein, an "individual" or "subject" is a mammal. Mammals include primates (e.g., humans and non-human primates such as monkeys) and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. The "subject" may be a "patient," which is a human subject in need of treatment, and may be an individual suffering from a CHRNA 7-related disease, a subject at risk of developing a CHRNA 7-related disease.

In some embodiments, the corresponding pharmaceutically acceptable salt refers to a salt of a compound of the present application that is substantially non-toxic to living organisms, e.g., a salt with an acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or a salt with a base such as sodium hydroxide, potassium hydroxide, or the like.

In some embodiments, the solvate refers to a combination of a compound of the present application formed by solvation with a solvent molecule, such as a combination of a compound with water to form a hydrate.

In some embodiments, the polymorphs refer to compounds of the present application that exist in different lattice forms.

In some embodiments, the composition may be administered by intravenous injection, oral administration, intramuscular administration, subcutaneous administration, topical administration, or the like, and the dosage forms thereof may be, but are not limited to, injection solutions, lyophilized powder for injection, injectable microspheres, liposomes, tablets, bilayer/multilayer tablets, buccal tablets, sublingual tablets, capsules, aqueous solutions, powders, pastes, sprays, granules, soft capsules, drop pills, gels, patches, ointments, and the like, wherein injection solutions, lyophilized powder for injection, tablets, and capsules are preferred.

Examples

The experimental methods in the following examples are conventional methods unless otherwise specified. The present application will be further understood with reference to the following non-limiting experimental examples.

EXAMPLE 1 discovery of CHRNA7 ligand

1. Compound data sources:

the Pubchem database CHRNA7 has been experimentally tested for 545 ligands with the inhibition constant Ki; searching compound component information of rhizoma atractylodis through a TCMSP database, and screening the compound component information through the biological oral availability (OB) of more than 30% to obtain 29 compound information; the PDB database was selected to have a CHRNA7 protein crystal structure of 5AFN.

2. Compound molecular descriptors calculated by open source semiochemical tools Rdkit and morred, including Rdkit molecular descriptors, ECFP molecular fingerprints, MACCS molecular fingerprints, and morred molecular descriptors.

3. The model of CHRNA7 inhibitory compounds classification was established by means of the machine model XGBoost, and after training the model super parameters were n_simulator=200, learning_rate=0.1, max_depth=5, random_state=0. The average accuracy of model classification can reach 89%, and the average AUC in the ROC curve of 10-fold cross test is 0.92. The method comprises the steps of screening rhizoma Atractylodis compounds through a model, specifically referring to figures 1-4, evaluating accuracy and stability of model prediction results through confusion matrix, ROC curve and ten-fold cross test, and finally carrying the rhizoma Atractylodis compounds into the model for prediction, wherein the results are shown in Table 1, and activity is 1 as an active molecule.

TABLE 1 classification of compounds in Atractylodes lancea by XGBoost training of the resulting model

4. According to the principle of structure-activity relationship, the structure and biological activity of the compound have great correlation. The potential active ingredients in rhizoma atractylodis are explored by molecular similarity calculation in the study. The molecular similarity is calculated using Tanimoto coefficients (also known as valley coefficients). The rhizoma Atractylodis compound is converted into RDKFINGERPRINT by Rdket, and similarity calculation is performed. The threshold was set at 50%, and results of the study on the similarity of specific rhizoma Atractylodis compounds to the CHRNA7 ligand Nicotine, BTX-a-comparison of the similarity of compounds to Nicotine after machine learning screening are shown in fig. 5,6, and table 2.

TABLE 2 selection of compounds with higher similarity to Nicotine, BTX-A by molecular similarity calculation

5. The CHRNA7 protein selected in the PDB database has a crystal structure of 5AFN, agonist binding site which is an agonist binding site and also an orthosteric binding site, and is bound by competitive inhibitors (ref: spurn R, debaveye S, farinha A, veys K, vos AM, gossas T, atack J, bertrand S, bertrand D, danielson UH, tresadern G, ulens C.molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the. Alpha 7nicotinic acetylcholine receptor.Proc Natl Acad Sci U S A.2015May 12;112 (19): E2543-52.doi:10.1073/pnas.1418289112.Epub 2015Apr 27.PMID:25918415;PMCID:PMC4434711.). Batch molecular docking of the atractylis compound components by autodock vina (see fig. 7), selecting small molecules with docking score less than-7, and obtaining 3 compound molecules: atractylone I (atractylenolide I), atractylone (atractyl one) and 3 beta-acetoxyatractylone (3 beta-acetoxyatractylone).

TABLE 3 molecular Butt screening by AutodockVina for higher Butt scoring molecules

6. The electrostatic potential of the ligand compound is calculated by ORAC quantum chemistry software, RESP charge data and topoisomers of the ligand compound are obtained, and molecular dynamics simulation is carried out by Gromacs. Setting the simulation time to be 100ns, selecting an AMBER99SB-ILDN protein force field parameterized protein receptor, and generating a topology file and a structure file. Molecular dynamics simulation results RMSD, RMSF, gyrate extraction it can be seen that three compound molecules reached structural stability after 100ns of simulation (see fig. 8).

7. Free energy profiles can be calculated from RMSD, gyrate results (see fig. 8), which can more intuitively show the stable conformation of the protein ligand complex at the energy nadir, PLIP calculates the interaction forces between complexes, where MOL000188 can form interactions with more amino acids, and MOL000164 can form pi-pi interactions.

8. Through virtual screening, see fig. 9, which shows the simulation results (lowest energy conformation and chemical bond) of the molecular dynamics of the atractyl compound and the CHRNA7, wherein a is the stable conformation of the MOL000043 and the CHRNA7 complex at the lowest free energy point, B is the stable conformation of the MOL000164 and the CHRNA7 complex at the lowest free energy point, C is the stable conformation of the MOL000188 and the CHRNA7 complex at the lowest free energy point, it can be seen that both atractylone and 3 β -acetoxyatractylone can form a relatively stable structure with CHRNA7, and the action site is the normal binding site of CHRNA7, possibly with competitive inhibition on the formation of CHRNA7 ligand, and may be a potential therapeutic drug or lead compound for various CHRNA 7-related diseases.

Example 2 verification of CHRNA7 ligand

To verify the regulatory effect of CHRNA7 on atractylone or 3 β -acetoxyatractylone, the present application verifies the affinity of nicotinic cholinergic receptor α7 (CHRNA 7) protein to atractylone. Taking atractylone as an example, in this example, affinity of CHRNa7 protein and atractylone molecule was evaluated by using Biacore8K analysis system (CM 5 chip as sensing chip), and sample information is shown in the following table:

the specific experimental scheme is as follows:

reagent preparation: 1. running Buffer:1 XPBS (2 mM KH) ₂ PO ₄ ,10mM Na ₂ HPO ₄ 137mM NaCl,2.7mM KCl) with 5% DMSO, pH7.4. 2. Regeneration Buffer:5mM NaOH,Immobilization Buffer 10mM Acetate,pH5.0 (BR 100351, GE). 3. Amine Coupling Kit (BR 100050, GE) which comprises 115mg of N-Hydroxysuccinimide (NHS), 750mg of 1-Ethyl-3- (3-dimethyllaminopyyl) carbodiimide hydrochloride (EDC) and 10.5mL 1M Ethanolamine hydrochloride-NaOH, pH8.5.EDC and NHS were dissolved by adding 10mL deionized water.

Chip preparation: 1. mix 400mM EDC and 100mM NHS (GE) to prepare activator, set the reagent flow rate to 10. Mu.L/min, and activate CM5 chip for 420 seconds. CHRNa7 at 50. Mu.g/mL was added with 10mM NaAc (pH 4.0) and the Fc4 sample channel was injected at a flow rate of 10. Mu.L/min to a fixed level of 5000 RU. The chip was blocked by 1M Ethanolamine hydrochloride-NaOH (GE) treatment at a flow rate of 10. Mu.L/min for 420 seconds. The reference surface Fc3 channel was blocked by the same procedure as Fc4, but without the step of ligand and CHRNa7 injection.

Analysis was run by a multi-cycle method: atractylone was diluted to 7 concentrations (100,50,25,12.5,6.25,3.125 and 0. Mu.M) with Running Buffer, injected into Fc3-Fc4 of the channel at a flow rate of 30. Mu.L/min, reacted for 60 seconds, followed by dissociation for 90 seconds. The association reaction and dissociation process are processed in a Running Buffer. The 6 cycles were repeated in ascending order according to the analyte concentration.

A CM5 chip was used to immobilize a quantitative amount of nicotinic cholinergic receptor alpha 7 (CHRNa 7), analyte was atractylone at a 6 concentration gradient, zero concentration was set with the reference channel, and solvent correction was performed. The analysis results are shown in Table 4 and FIG. 10.

TABLE 4 affinity of CHRNa7 protein to atractylone

It can be seen that nicotinic cholinergic receptor alpha 7 (CHRNa 7) and atractylone have high affinity, and atractylone is an effective ligand of CHRNa7, and can bind to CHRNa7 in vitro and in vivo, so that the function of the nicotinic cholinergic receptor alpha 7 can be regulated and the nicotinic acid can be applied to the field of medicine.

The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the present application and its practical application to thereby enable one skilled in the art to make and utilize the present application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and the equivalents thereof.

Claims (9)

1. A method for obtaining a CHRNA7 ligand by letter screening, comprising the steps of:

1) Modeling a test ligand of CHRNA7 in a Pubchem database by using a machine learning model XGBoost; 2) Screening the compound contained in the rhizoma atractylodis in the TCMSP database by using the established classification model, molecular similarity and molecular docking;

3) Using molecular dynamics to simulate the binding mode and binding situation of the compound and CHRNA 7;

4) Verification was performed based on SPR affinity experiments.

2. The method of claim 1, wherein the chra 7 ligand is atractylone or 3β -acetoxyatractylone.

3. A method of modulating a CHRNA7 protein, comprising the step of administering atractylone or 3 β -acetoxyatractylone to a sample.

4. A method according to claim 3, wherein the sample is an in vivo or in vitro sample.

5. The method of claim 3, wherein the modulation is inhibition or reduction of chra 7 protein activity or function; the inhibition or reduction is achieved by binding atractylone or 3 beta-acetoxyatractylone to the CHRNA7 protein.

6. Any one of the following applications of atractylone or 3 beta-acetoxyatractylone:

a) Use as a chra 7 ligand;

b) Use in modulating CHRNA 7;

c) The application in preparing a preparation for regulating and controlling CHRNA 7;

d) The application in preparing medicines for treating and/or preventing CHRNA7 related diseases;

e) Use in screening and/or identifying candidate inhibitors of CHRNA 7-related diseases.

7. The use according to claim 6, wherein the modulation is inhibition or reduction of chra 7 protein activity or function; the inhibition or reduction is achieved by binding atractylone or 3 beta-acetoxyatractylone to the CHRNA7 protein.

8. A method of screening and/or identifying candidate inhibitors, comprising any of the steps of:

a) Analyzing the effect of the compound on the binding of atractylone or 3 beta-acetoxyatractylone to the receptor CHRNA7 to obtain a candidate inhibitor;

b) Screening compounds for inhibiting interaction of atractylone or 3 beta-acetoxyatractylone and CHRNA7 to obtain candidate inhibitors;

the inhibitor is selected from a small molecule inhibitor, a polypeptide inhibitor or an antibody inhibitor.

9. A pharmaceutical composition characterized by: the composition comprisesLigands for CHRNA7And a pharmaceutically acceptable carrier or excipient; the saidLigands for CHRNA7Is atractylone or 3 beta-acetoxyatractylone.