Screening method of small molecule inhibitor of COVID-19 spinous process protein, active molecule screened by same and application
阅读说明：本技术 一种covid-19棘突状蛋白的小分子抑制剂的筛选方法、其筛选的活性分子及用途 (Screening method of small molecule inhibitor of COVID-19 spinous process protein, active molecule screened by same and application ) 是由 李悦青 赵伟杰 潘霞 严梦雪 孙萌萌 于 2020-12-29 设计创作，主要内容包括：本发明公开了一种COVID-19棘突状蛋白抑制剂的筛选方法、其筛选的活性分子及用途,所述的筛选方法从数据库获取COVID-19棘突状蛋白与人血管紧张素转化酶2共晶的三维结构；从中药材活性成分数据库获取中药材的活性分子及其衍生物,构建小分子的化合物库；建立药效团模型,将药效团模型作为查询式,基于匹配度值筛选,选取归一化匹配值大于0.35的小分子化合物。本发明可以实现针对新冠病毒侵入人体细胞的S蛋白结合人ACE2环节、快速有效的筛选出有效的S蛋白小分子抑制剂,所筛出的抑制剂效果确切,在预防或/和治疗新型冠状病毒肺炎上具有应用前景。(The invention discloses a screening method of a COVID-19 spinous protein inhibitor, an active molecule screened by the screening method and application, wherein the screening method obtains a three-dimensional structure of cocrystallization of the COVID-19 spinous protein and human angiotensin converting enzyme 2 from a database; obtaining active molecules and derivatives thereof of the traditional Chinese medicinal materials from a traditional Chinese medicinal material active ingredient database, and constructing a small molecular compound library; and establishing a pharmacophore model, taking the pharmacophore model as an inquiry formula, screening based on the matching degree value, and selecting the micromolecular compound with the normalized matching value larger than 0.35. The invention can realize the combination of the S protein of the new coronavirus invading human body cell and human ACE2 link, quickly and effectively screen out the effective S protein small molecular inhibitor, the screened inhibitor has exact effect, and has application prospect in preventing or/and treating the novel coronavirus pneumonia.)
1. A screening method of a small molecule inhibitor of COVID-19 spinous process protein is characterized by comprising the following steps:
step 1, obtaining a three-dimensional structure of cocrystallization of COVID-19 spinous process protein and human angiotensin converting enzyme 2 from a database;
step 2, obtaining active molecules and derivatives of the traditional Chinese medicinal materials from a traditional Chinese medicinal material active ingredient database, and constructing a small molecular compound library;
step 3, establishing a pharmacophore model based on the binding characteristics of the human angiotensin converting enzyme 2 and the COVID-19 spinous process protein as a target protein by using the hydrogen bond interaction of the COVID-19 spinous process protein and the human angiotensin converting enzyme 2 in the three-dimensional structure obtained in the step 1), wherein the pharmacophore model is manually generated by using Discovery Studio based on the 24 th amino acid Gln24, the 30 th amino acid Asp30, the 34 th amino acid His34, the 42 th amino acid Gln42, the 83 th amino acid Tyr83, the 353 th amino acid Lys353 and the 355 th amino acid Asp355 of the human angiotensin converting enzyme 2, and comprises two hydrogen bond donor characteristics and six hydrogen bond acceptor characteristics;
and 4) adopting the pharmacophore model constructed in the step 3) as an inquiry formula, screening the micromolecule compound library constructed in the step 2) based on the matching value, and selecting the micromolecule compound with the normalized matching value larger than 0.35 as a potential micromolecule inhibitor of the COVID-19 spinous process protein.
2. The method of screening for small molecule inhibitors of COVID-19 spinous process proteins of claim 1, wherein in step 3), the two hydrogen bond donor features and six hydrogen bond acceptor features of the pharmacophore model are specifically:
based on the fact that an amide nitrogen atom of Gln24 is used as a characteristic point center of a hydrogen bond donor pharmacophore, and an amide carbonyl oxygen of Asn487 of hACE2, which forms a hydrogen bond with the amide nitrogen atom, is used as another characteristic point center, the hydrogen bond donor comprising two characteristic points and a vector direction is established;
based on that the carboxylic acid carbonyl oxygen of Asp30 is the center of a characteristic point of a pharmacophore of a hydrogen bond receptor, and the amino nitrogen of Lys417 of hACE2 which forms a hydrogen bond with the carboxylic acid carbonyl oxygen is used as the center of another characteristic point, establishing the hydrogen bond receptor which comprises two characteristic points and a vector direction;
based on the fact that imidazole aromatic nitrogen of His34 is used as a characteristic point center of a hydrogen bond receptor pharmacophore, hydroxyl oxygen of Tyr453 of hACE2 which forms a hydrogen bond with the imidazole aromatic nitrogen is used as another characteristic point center, and a hydrogen bond receptor comprising two characteristic points and a vector direction is established;
based on that amide carbonyl oxygen of Gln42 is a characteristic point center of a hydrogen bond receptor pharmacophore, hydroxyl oxygen of Tyr449 of hACE2 which forms a hydrogen bond with the amide carbonyl oxygen is another characteristic point center, and a hydrogen bond receptor comprising two characteristic points and a vector direction is established;
based on that the hydroxyl oxygen of Tyr83 is the center of a characteristic point of a pharmacophore of a hydrogen bond receptor, the middle point of the amino nitrogen of Asn487 of hACE2 and the hydroxyl oxygen of Tyr489 which form a hydrogen bond with the pharmacophore is taken as the center of another characteristic point, and the hydrogen bond receptor comprising two characteristic points and a vector direction is established;
establishing a hydrogen bond receptor comprising two characteristic points and a vector direction by taking carbonyl oxygen of Lys353 as a characteristic point center of a hydrogen bond receptor pharmacophore and taking amino nitrogen of Gly502 of hACE2 which is in hydrogen bond with the carbonyl oxygen as another characteristic point center;
establishing a hydrogen bond donor comprising two characteristic points and a vector direction by taking the amino nitrogen of Lys353 as a characteristic point center of a hydrogen bond donor pharmacophore and the carbonyl oxygen of Gly496 of hACE2 which is in hydrogen bond with the amino nitrogen of Lys353 as another characteristic point center;
the carbonyl oxygen of carboxylic acid based on Lys355 is the center of a characteristic point of a pharmacophore of a hydrogen bond acceptor, and the hydroxyl oxygen of Thr500 of hACE2 which is hydrogen bonded with the carbonyl oxygen is the center of another characteristic point, so that the hydrogen bond acceptor comprising two characteristic points and a vector direction is established.
3. The method for screening small molecule inhibitors of COVID-19 spinous process proteins of claim 1 or 2, wherein in step 4), the screening is performed using Ligand Pharmacophore Mapping under the Pharmacophores module in the software Discovery Studio.
4. The method for screening the small molecule inhibitor of COVID-19 spinous process protein of any one of claims 1 to 3, wherein in step 4), the small molecule compound with the normalized matching value of more than 0.39 is selected as the potential small molecule inhibitor of COVID-19 spinous process protein; more preferably, the small molecule compound with the normalized matching value larger than 0.4 is selected as a potential small molecule inhibitor of the COVID-19 spinous process protein.
5. The method for screening a small molecule inhibitor of COVID-19 spinous process protein of any one of claims 1-4, further comprising, after the screening of step 4):
and 5) verifying the potential small molecule inhibitor of the COVID-19 spinous process protein obtained in the step 4).
6. Use of a small molecule inhibitor obtained by the screening method for a small molecule inhibitor of COVID-19 spinous process protein according to any one of claims 1-5 in the manufacture of an agent for treating and/or preventing a disease caused by a novel coronavirus infection, wherein the small molecule inhibitor is selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157 and combinations thereof.
7. The use according to claim 6, wherein the disease caused by the novel coronavirus infection is novel coronavirus pneumonia;
preferably, the novel coronavirus pneumonia is novel coronavirus pneumonia in a mammal;
more preferably, the mammal is a human.
8. The use according to claim 6 or 7, wherein the agent is for the treatment and/or prevention of diseases caused by a novel coronavirus infection by blocking the binding of COVID-19spike protein to human angiotensin converting enzyme 2.
9. The use according to claim 6 or 7, wherein the agent for preventing diseases caused by the novel coronavirus infection is a hand sanitizer, a mouth wash or a nasal spray.
10. A pharmaceutical composition comprising a compound selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157 and combinations thereof; the pharmaceutical composition is used for treating and/or preventing diseases caused by the infection of the novel coronavirus.
Technical Field
The invention belongs to the technical field of medicine and biology, and particularly relates to a screening method of a COVID-19 spinous process protein inhibitor, an active molecule screened by the inhibitor and application of the inhibitor.
Background
Human coronavirus Spike protein (S protein) is a kind of virus fusion protein, and the binding of S protein to host receptor is a key step of virus infection of cells and can be used as a target of drugs for inhibiting human coronavirus [ Structural and functional properties of SARS-CoV-2 Spike protein: potential anti-virus drug definition for COVID-19, Acta Pharmacological site, 2020 ]. In the month 4, the crystal structure (PDB ID: 6LZG) of the complex of the S protein of COVID-19 and angiotensin-converting enzyme 2(ACE2) on the surface of human cells was analyzed by the cooperation of multiple groups of scientists in China. The human novel coronavirus COVID-19 belongs to a beta coronavirus, and the S1 subunit of the S protein of the coronavirus contains a plurality of structural domains A-D, wherein the structural domains A and B form a Receptor domain (RBD) and interact with human angiotensin converting enzyme 2(hACE2, human ACE 2). At present, the research aiming at the target spot is mainly carried out on the design of host neutralizing antibodies and vaccines, and the reports of small molecule inhibitors are rare.
In China, traditional Chinese medicine plays an important role in treatment and prevention and control of novel coronavirus, and in multiple versions of diagnosis and treatment scheme (trial version) for novel coronavirus pneumonia issued by the national Wei-Jian Commission, Chinese patent medicine and traditional Chinese medicine prescriptions are all written into the Chinese medicine and the Chinese medicine plays a great role in clinical treatment. Some current studies of papers have also gradually confirmed the role of active ingredients in traditional Chinese medicines in various links of human body infected by virus and causing inflammatory reaction, for example, the southern mountain academy team published on Pharmacological Research under the name of Lianhuaqingwen exirts anti-viral and anti-inflammatory activity against novel coronaviruses (SARS-CoV-2), which shows that the Lianhuaqingwen can significantly inhibit the replication of the novel coronaviruses (SARS-CoV-2) in cells, thereby playing the role of anti-new coronaviruses activity.
With the development of computer-aided drug design, virtual drug screening has become a technology complementary to high-throughput screening, and is an important method for finding lead compounds and optimizing the activity thereof. Therefore, a quick and efficient screening method for inhibiting the COVID-19 spinous process protein small molecules is established, and the method has important significance for the discovery of potential and definite-action COVID-19 spinous process protein inhibition and the application of the inhibition in the prevention and treatment of novel coronaviruses.
Disclosure of Invention
The invention aims to provide a screening method of a high-efficiency COVID-19 spinous process protein small molecule inhibitor based on virtual screening and application of a target compound in prevention and treatment of novel coronavirus.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a screening method of a small molecule inhibitor of COVID-19 spinous process protein, which comprises the following steps:
step 1, obtaining a three-dimensional structure of cocrystallization of COVID-19 spinous process protein and human angiotensin converting enzyme 2 from a database;
step 2, obtaining active molecules and derivatives of the traditional Chinese medicinal materials from a traditional Chinese medicinal material active ingredient database, and constructing a small molecular compound library;
step 3, establishing a pharmacophore model based on the binding characteristics of the human angiotensin converting enzyme 2 and the COVID-19 spinous process protein as a target protein by using the hydrogen bond interaction of the COVID-19 spinous process protein and the human angiotensin converting enzyme 2 in the three-dimensional structure obtained in the step 1), wherein the pharmacophore model is manually generated by using Discovery Studio based on the 24 th amino acid Gln24, the 30 th amino acid Asp30, the 34 th amino acid His34, the 42 th amino acid Gln42, the 83 th amino acid Tyr83, the 353 th amino acid Lys353 and the 355 th amino acid Asp355 of the human angiotensin converting enzyme 2, and comprises two hydrogen bond donor characteristics and six hydrogen bond acceptor characteristics;
and 4) adopting the pharmacophore model constructed in the step 3) as an inquiry formula, screening the micromolecule compound library constructed in the step 2) based on the matching value, and selecting the micromolecule compound with the normalized matching value larger than 0.35 as a potential micromolecule inhibitor of the COVID-19 spinous process protein.
In a specific embodiment, in step 1), the database is a Protein data bank, and the accession number of the three-dimensional structure of cocvid-19 spinous process Protein and human angiotensin converting enzyme 2 cocrystal is PDB ID: 6 LZG.
In a specific embodiment, in step 2), the Chinese medicinal material active ingredient Database is selected from TCMSP (Traditional Chinese Medicine Systems pharmacy Database and Analysis Platform, https:// tcmspw.com/TCMSP. php) and TCMID (Traditional Chinese Medicine Integrated Database, http:// www.megabionet.org/TCMID).
Further, the Chinese medicinal materials include but are not limited to: fructus forsythiae, honeysuckle, bitter apricot seed, indigowoad root, heartleaf houttuynia herb, cablin potchouli herb, rhubarb, liquorice, ephedra herb, rhodiola rosea, rhizoma dryopteris crassirhizomae, rhizoma atractylodis, dried orange peel, officinal magnolia bark, dahurian angelica root, Indian buead, areca peel, pinellia tuber, liquorice extract, perilla leaf oil, oriental waterplantain rhizome, umbellate pore fungus, almond, cassia twig, largehead atractylodes rhizome, radix bupleuri, baical skullcap root, ginger, aster, common coltsfoot flower, blackberry lily, asarum, Chinese yam, immature bitter orange, dahurian patrinia herb, giant knotweed rhizome, verbena.
In a specific embodiment, in step 3), the two hydrogen bond donor characteristics and the six hydrogen bond acceptor characteristics of the pharmacophore model are specifically:
based on the fact that an amide nitrogen atom of Gln24 is used as a characteristic point center of a hydrogen bond donor pharmacophore, and an amide carbonyl oxygen of Asn487 of hACE2, which forms a hydrogen bond with the amide nitrogen atom, is used as another characteristic point center, the hydrogen bond donor comprising two characteristic points and a vector direction is established;
based on that the carboxylic acid carbonyl oxygen of Asp30 is the center of a characteristic point of a pharmacophore of a hydrogen bond receptor, and the amino nitrogen of Lys417 of hACE2 which forms a hydrogen bond with the carboxylic acid carbonyl oxygen is used as the center of another characteristic point, establishing the hydrogen bond receptor which comprises two characteristic points and a vector direction;
based on the fact that imidazole aromatic nitrogen of His34 is used as a characteristic point center of a hydrogen bond receptor pharmacophore, hydroxyl oxygen of Tyr453 of hACE2 which forms a hydrogen bond with the imidazole aromatic nitrogen is used as another characteristic point center, and a hydrogen bond receptor comprising two characteristic points and a vector direction is established;
based on that amide carbonyl oxygen of Gln42 is a characteristic point center of a hydrogen bond receptor pharmacophore, hydroxyl oxygen of Tyr449 of hACE2 which forms a hydrogen bond with the amide carbonyl oxygen is another characteristic point center, and a hydrogen bond receptor comprising two characteristic points and a vector direction is established;
based on that the hydroxyl oxygen of Tyr83 is the center of a characteristic point of a pharmacophore of a hydrogen bond receptor, the middle point of the amino nitrogen of Asn487 of hACE2 and the hydroxyl oxygen of Tyr489 which form a hydrogen bond with the pharmacophore is taken as the center of another characteristic point, and the hydrogen bond receptor comprising two characteristic points and a vector direction is established;
establishing a hydrogen bond receptor comprising two characteristic points and a vector direction by taking carbonyl oxygen of Lys353 as a characteristic point center of a hydrogen bond receptor pharmacophore and taking amino nitrogen of Gly502 of hACE2 which is in hydrogen bond with the carbonyl oxygen as another characteristic point center;
establishing a hydrogen bond donor comprising two characteristic points and a vector direction by taking the amino nitrogen of Lys353 as a characteristic point center of a hydrogen bond donor pharmacophore and the carbonyl oxygen of Gly496 of hACE2 which is in hydrogen bond with the amino nitrogen of Lys353 as another characteristic point center;
the carbonyl oxygen of carboxylic acid based on Lys355 is the center of a characteristic point of a pharmacophore of a hydrogen bond acceptor, and the hydroxyl oxygen of Thr500 of hACE2 which is hydrogen bonded with the carbonyl oxygen is the center of another characteristic point, so that the hydrogen bond acceptor comprising two characteristic points and a vector direction is established.
Furthermore, the amino acid sequence of the human angiotensin converting enzyme 2 is shown in SEQ ID NO 1.
In a specific embodiment, in step 4), the screening is performed using Ligand Pharmacophore Mapping under the Pharmacophores module in the software Discovery Studio.
Preferably, in the step 4), selecting a small molecule compound with a normalized matching value larger than 0.39 as a potential small molecule inhibitor of the COVID-19 spinous process protein; more preferably, the small molecule compound with the normalized matching value larger than 0.4 is selected as a potential small molecule inhibitor of the COVID-19 spinous process protein.
In an alternative embodiment, after the screening in step 4), the method further comprises:
and 5) verifying the potential small molecule inhibitor of the COVID-19 spinous process protein obtained in the step 4).
In a second aspect, the present invention provides a use of a small molecule inhibitor obtained by the screening method of the small molecule inhibitor of COVID-19 spinous process protein described above in the preparation of an agent for treating and/or preventing a disease caused by a novel coronavirus infection, wherein the small molecule inhibitor is selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157, herbal extracts comprising the screened small molecules, and combinations thereof.
Further, the disease caused by the novel coronavirus infection is novel coronavirus pneumonia.
Further, the novel coronavirus pneumonia is novel coronavirus pneumonia in mammals; preferably, the mammal is a human.
Further, the agent treats and/or prevents diseases caused by the novel coronavirus infection by blocking the binding of COVID-19 spinous process protein and human angiotensin converting enzyme 2.
Further, the agent for preventing diseases caused by the novel coronavirus infection is a hand sanitizer, a mouth wash or a nasal spray.
In a third aspect, the present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157 and combinations thereof; the pharmaceutical composition is used for treating and/or preventing diseases caused by the infection of the novel coronavirus.
In a fourth aspect, a method of blocking binding of COVID-19 spinous process protein to human angiotensin converting enzyme 2 comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157 and combinations thereof.
In a fifth aspect, the present invention provides a method of treating a disease caused by a novel coronavirus infection, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157 and combinations thereof.
In a sixth aspect, the present invention provides a method of blocking the binding of COVID-19 spinous process protein to human angiotensin converting enzyme 2, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of: small molecule compounds numbered DUT202009 to DUT202157, and combinations thereof.
The invention has the advantages that:
according to the invention, the screening efficiency of the target COVID-19 spinous protein small molecule inhibitor is improved by manually constructing a pharmacophore model based on experience and setting a mode of reasonable pharmacophore screening parameters, and the virtual screening time of each molecule is less than 1 minute; the enrichment rate of active molecules is improved, and the molecules with the FitValue value of more than 0.35 only account for 10 percent of the screened molecules; through the combined transcription detection experiment of the COVID-19S protein viroid and human ACE2 on the cell surface and the viroid cell detection verification of the novel coronavirus S protein, the target compound obtained by calculation and screening really plays an obvious blocking role in the mutual combination of the COVID-19S protein viroid and the human ACE2 on the cell surface. The invention can realize the combination of the S protein of the new coronavirus invading human body cell and human ACE2 link, quickly and effectively screen out the effective S protein small molecular inhibitor, the screened inhibitor has exact effect, and has application prospect in preventing or/and treating the novel coronavirus pneumonia.
Drawings
FIG. 1 shows amino acid residues and hydrogen bonds on the interaction interface of the COVID-19S protein and human ACE 2.
Fig. 2 is a virtual screening pharmacophore model established based on key residues of human ACE 2.
FIG. 3 is a graph of the inhibition of the binding of fluorescently labeled COVID-19 spinous process protein and cells expressing fluorescently labeled human angiotensin converting enzyme 2 by 100 μ M compound DUT202001 under a 100-fold microscope.
FIGS. 4A to 4C are graphs showing the results of a viroid cell assay system based on the S protein of the novel coronavirus, wherein FIG. 4A shows the assay results of compound DUT202001, FIG. 4B shows the assay results of compound DUT202003, and FIG. 4C shows the assay results of compound DUT 202004.
Detailed Description
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Term (definition)
In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
In the present specification, the term "small molecule compound" refers to: relative molecular mass not greater than 1000, and is not a compound of small peptides, oligopeptides, oligosaccharides and oligonucleotides.
The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking when ligand binding occurs.
By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the compounds of the present invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in each patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any of the statistical tests known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.
"pharmaceutical composition" means a mixture containing one or more compounds described in the present disclosure, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
An "effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening severity, or delaying onset of the condition, including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes exhibited during development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of a disease caused by various novel coronavirus infections of the present disclosure, reducing the dosage of another agent required to treat a condition, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a disease caused by a novel coronavirus infection in a patient.
The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivery of the antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antibacterial or antifungal agent), sweetener, absorption retarder, wetting agent, emulsifier, buffer, or the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, dextrose in polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.
An "effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening severity, or delaying onset of the condition, including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes exhibited during development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-associated disorders of the disclosure, reducing the dosage of other agents required to treat the disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a target antigen-associated disorder of the disclosure in a patient.
"administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
Example (b): screening of COVID-19S protein inhibitor
In this example, the specific steps for screening the COVID-19S protein inhibitor are as follows:
1) the three-dimensional structure of the cocrystal of the COVID-19S Protein and the human ACE2 Protein is obtained from Protein data bank (PDB ID: 6 LZG).
2) Active molecules in traditional Chinese medicinal materials, namely fructus forsythiae, honeysuckle, bitter apricot seed, isatis root, houttuynia cordata, patchouli, rhubarb, liquorice, ephedra, rhodiola rosea, rhizoma dryopteris crassirhizomae, rhizoma atractylodis, dried orange peel, mangnolia officinalis, angelica dahurica, poria cocos, pericarpium arecae, pinellia ternate, liquorice extract, perilla leaf oil, rhizoma alismatis, grifola, almond, cassia twig, rhizoma atractylodis macrocephalae, radix bupleuri, scutellaria baicalensis, ginger, aster, flos farfarae, blackberry lily, asarum, yam, immature bitter orange, dahurian patrinia herb, polygonum cuspidatum, verbena, reed rhizome, thunberg fritillary bulb, burdock, sweet wormwood and veratrum nigrum, and derivatives of partial active molecules are downloaded from a traditional Chinese medicinal material active ingredient database (TCMSP, TCMID and.
3) According to the three-dimensional structure of the cocrystal of the COVID-19S protein and the human ACE2 protein obtained in the step 1), based on the hydrogen bond interaction of the COVID-19S protein and the human ACE2 protein, the S protein of the COVID-19 is used as a target protein, and a pharmacophore model is established based on the characteristic that the human ACE2 protein and the S protein are combined.
Specifically, in this example, a pharmacophore model was generated manually using Discovery Studio based on amino acid Gln24 at position 24, amino acid Asp30 at position 30, amino acid His34 at position 34, amino acid Gln42 at position 42, amino acid Tyr83 at position 83, amino acid Lys353 at position 353, and amino acid Asp355 of human ACE2 protein in a three-dimensional structure of cocvid-19S protein and human ACE2 eutectic. The pharmacophore model comprises two hydrogen bond donor characteristics and six hydrogen bond acceptor characteristics, and comprises pharmacodynamic characteristic elements of key hydrogen bond donor-acceptor relation formed by combination of human ACE2 and COVID-19, as shown in figure 2.
Based on that the amide nitrogen atom of Gln24 is a characteristic point center (-35.415, 48.299, -2.863; radius is 1.6 angstroms) of a hydrogen bond donor pharmacophore, the amide carbonyl oxygen of Asn487 of hACE2 which forms a hydrogen bond with the amide nitrogen atom is taken as another characteristic point center (-38.2783, 47.4277, -2.65727, radius is 2.2 angstroms), and the hydrogen bond donor which comprises two characteristic points and one vector direction is established; based on that the carboxylic acid carbonyl oxygen of Asp30 is the center (-30.831, 36.305, 3.58; radius 1.6 angstrom) of a characteristic point of a pharmacophore of a hydrogen bond receptor, and the amino nitrogen of Lys417 of hACE2 which forms a hydrogen bond with the carboxylic acid carbonyl oxygen is the center (-30.823, 35.4579, 6.4579; radius 2.2 angstrom) of another characteristic point, a hydrogen bond receptor comprising two characteristic points and a vector direction is established; based on that imidazole aromatic nitrogen of His34 is a characteristic point center (-32.54, 30.603, 4.172; radius 1.6 angstroms) of a hydrogen bond receptor pharmacophore, hydroxyl oxygen of Tyr453 of hACE2 which forms a hydrogen bond with the imidazole aromatic nitrogen is used as another characteristic point center (-35.1122, 29.3297, 6.17518; radius 2.2 angstroms), and a hydrogen bond receptor comprising two characteristic points and a vector direction is established; amide carbonyl oxygen based on Gln42 is a characteristic point center (-42.888, 19.226, 0.156; radius 1.6 angstroms) of a pharmacophore of a hydrogen bond receptor, hydroxyl oxygen of Tyr449 of hACE2 which forms a hydrogen bond with the amide carbonyl oxygen is another characteristic point center (-43.2949, 21.4028, 2.17987; radius 2.2 angstroms), and the hydrogen bond receptor which comprises two characteristic points and a vector direction is established; based on that the hydroxyl oxygen of Tyr83 is a characteristic point center (-36.944, 43.244, -3.855; radius is 1.6 angstroms) of a hydrogen bond acceptor pharmacophore, the middle point of the amino nitrogen of Asn487 of hACE2 and the hydroxyl oxygen of Tyr489 which are in hydrogen bond with the hydroxyl oxygen is another characteristic point center (-36.0547, 42.6713, -1.04766; radius is 2.2 angstroms), and the hydrogen bond acceptor comprising two characteristic points and one vector direction is established; based on that carbonyl oxygen of Lys353 is a characteristic point center (-31.489, 17.337, 1.754; radius 1.6 angstroms) of a pharmacophore of a hydrogen bond receptor, and amino nitrogen of Gly502 of hACE2 which forms a hydrogen bond with the carbonyl oxygen is another characteristic point center (-31.5854, 15.0581, 3.70273; radius 2.2 angstroms), the hydrogen bond receptor comprising two characteristic points and a vector direction is established; based on that the amino nitrogen of Lys353 is taken as a characteristic point center (-36.301, 21.41, 3.197; radius 1.6 angstroms) of a hydrogen bond donor pharmacophore, and the carbonyl oxygen of Gly496 of hACE2 which is in hydrogen bond with the amino nitrogen is taken as another characteristic point center (-38.1973, 19.7902, 4.86432; radius 2.2 angstroms), the hydrogen bond donor comprising two characteristic points and one vector direction is established; the carbonyl oxygen of carboxylic acid based on Lys355 is the center of characteristic point of pharmacophore of hydrogen bond acceptor (-34.593, 13.071, -1.534; radius 1.6 angstrom), and the hydroxyl oxygen of Thr500 of hACE2 which is hydrogen bonded with the carbonyl oxygen is the center of another characteristic point (-37.1642, 12.5286, 0.777888; radius 2.2 angstrom), so as to establish the hydrogen bond acceptor which comprises two characteristic points and one vector direction.
Specifically, the amino acid sequence of the human ACE2 protein is shown below (SEQ ID NO: 1):
MSSSSWLLLS LVAVTAAQST IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEM ARANHYEDYG DYWRGDYEVNGVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKEAEKFFVSVGLPNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILMCTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP PHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYADQSIKV RISLKSALGD KAYEWNDNEM YLFRSSVAYA MRQYFLKVKN QMILFGEEDV RVANLKPRIS FNFFVTAPKN VSDIIPRTEV EKAIRMSRSR INDAFRLNDN SLEFLGIQPT LGPPNQPPVS IWLIVFGVVM GVIVVGIVIL IFTGIRDRKK KNKARSGENP YASIDISKGE NNPGFQNTDD VQTSF
4) screening the small molecule compound library in the step 2) based on the matching value by using the pharmacophore model generated in the step 3) as a query expression. Specifically, Ligard Pharmacophore Mapping under Pharmacophores module is used for screening, and the main parameters are set as follows:
formation Generation (Conformation Generation): FAST (Rapid)
Maximum Conformations: 255
Discard Existing configurations (elimination of Existing conformation): true (Yes)
Energy Threshold (Energy Threshold): 20.0
Best Mapping Only (retaining the Best match): true (Yes)
Options: fit most features diseases (the most matching pharmacodynamic characteristics)
Maximum ordered Features (Maximum negligible Features): -1
Fitting Method (matching Method): flexile (flexibility)
Minimum interface Distance (Minimum Distance between features): 0.5
Map Each transformation separator (Each Conformation matches individually): true (Yes)
Fit Name (match Name): FitValue (match value)
Scale Fit Values (normalized match Values): true (Yes)
5) Screening the 5521-containing small molecule compound library obtained in the step 2) by adopting the parameter setting in the step 4) to obtain a primary screening result containing 534 compounds;
6) in the preliminary screening result obtained in step 5), 257 small molecule compounds with normalized FitValue greater than 0.35, 69 of which have no specific CAS number, and 35 small molecule compounds have been reported in the literature to have resistance to diseases caused by novel coronavirus infection, which proves that the screening method adopted in this example is effective, for example, saikosaponin a (CAS number: 20736-09-8, numbered DUT202008 in the subsequent test examples, as a control), it was demonstrated in the published patent that it was possible to inhibit infection of ACE2 high expressing cells (CN111904971A) with COVID-19 pseudovirus, which in this example, has a FitValue of 0.4585.
Additional 153 small molecule compounds, see in particular table 1 below:
table 1: molecules with anti-neocorona activity are not reported when Fitvalue value obtained by screening is higher than 0.35
Test example 1: activity test for blocking the binding of the Receptor Binding Domain (RBD) of the CoVID-19 spinous Process protein to human angiotensin converting enzyme 2(hACE2)
Experimental materials: compounds DUT202001, DUT202002, DUT202003, DUT202004, and DUT202010 (laboratory owned); compounds DUT202008 and DUT202009 (available from wakaki biotechnology limited, sikawa); SARS-CoV-2S, pAX2, pHB and ACE2-pcDNA3 (Scopulars technologies, Inc.); 293T cells (Shanghai Synbiotic Biotech Co., Ltd.). The pCMV-SNAP-hACE2 plasmid, SNAP-561 fluorescent dye and RBD541-Halo-640dye were obtained from the Xumega ultra research group of the institute of chemico-physical, university of Chinese academy of sciences.
Specifically, the test procedure is as follows:
1) selecting part of the compounds in the table 1 in the above examples for activity detection, specifically, selecting luteolin disulfonated derivative MXCS-diSO with normalized FitValue score of 0.3966 as virtual screening3Na (DUT202001), luteolin disulfonated derivative MXCS-diSO with normalized FitValue score of 0.4463Na (DUT202002), resveratrol analogue with normalized FitValue score of 0.400 (DUT202003), veratrum alkaloid with normalized FitValue score of 0.38 (DUT202010), saikosaponin b2 with normalized FitValue score of 0.4626 (DUT202009), saikosaponin a with normalized FitValue score of 0.4585 (DUT202008 as positive control), starting at the respective appropriate concentrations, the tested compounds were selected as shown in table 2 below.
Table 2 test compound information
2) Experiment for blocking binding of Receptor Binding Domain (RBD) of COVID-19 spinous process protein and human angiotensin converting enzyme 2(hACE2)
Hela cells were transferred to 2 confocal imaging dishes and after 24 hours, transiently transferred to pCMV-SNAP-hACE2 plasmid with Lipo2000 reagent at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. Cells were incubated with the probe solution for 10min and DMEM was used once. The control group was supplemented with 1mL of DMEM medium containing 20nM RBD541-Halo-640dye alone, and the experimental group was supplemented with 1mL of DMEM medium containing 120. mu.M of the drug molecule to be tested and 20nM RBD541-Halo-640dye together. 5% CO at 37 ℃2Incubate in incubator for 60 min. Imaging was performed with a fluorescence confocal microscope under a 10-fold microscope, as shown in FIG. 3.
The imaging channel of RBD541-Halo protein excited at 640nM in FIG. 3 and the imaging channel of SNAP-ACE2 protein excited at 561 nM in the second behavior, wherein (a) in FIG. 3 is the imaging of cells with 20nM RBD541-Halo-640dye added only; FIG. 3 (b) is the cell image of the cell added with 20nM RBD541-Halo-640dye and 120. mu.M drug molecule to be tested DUT202001, the red channel has weak fluorescence, which shows that the molecule has the function of inhibiting the binding of SARS-CoV-2 RBD and hACE 2.
In addition, we counted the mean fluorescence intensity of 20 cellsAndand average fluorescence intensity of the background of the imaged pictureAnd at IF of (a) in FIG. 3640/IF561As the relative RBD Activity (RA) of 100%, fig. 3 (b) was compared with fig. 3 (a), and 120 μ M of the molecule was found to inhibit 85% of RBD activity.
The results of selected compound testing are shown in table 3.
TABLE 3 results of inhibiting the binding of COVID-19 spinous proteins to human angiotensin converting enzyme 2
According to the above experimental method, the results of activity assay of the selected compounds are shown in Table 3 and FIG. 3, and the candidate compounds have inhibitory effects on COVID-19 spinous process protein RBD and Hela cells expressing hACE2 to different degrees, especially compound DUT202001 at a concentration of 7.6. mu.M, which is 51.8%.
Test example 2: evaluation of viroid cell detection system based on luciferase reporter system and aiming at novel coronavirus S protein
In this test example, the viroid cell detection of the S protein of the novel coronavirus was performed by the science and technology company, Colpithei, Bio-medicine, Inc., in Hefei. Briefly, the method comprises the following steps:
1) preparing and collecting viroid of new coronavirus S protein: pseudovirus 293T cells were transiently transfected with three pseudovirus plasmids SARS-CoV-2S, pAX2 and pHB, the S protein as the envelope protein, and the luciferase gene packaged inside the virus. Changing fresh culture medium after 6-8h, at 37 deg.C and 5% CO2Culturing for 48 h. Viral supernatants were collected from 60mm dishes and filtered through 0.45um filters and used immediately or stored at-80 ℃.
2) Preparation of 293T cells expressing hACE2 on the surface and plating: transient transfection of 293T cells with ACE2-pcDNA3 cell plasmid 6-8h laterFresh medium was changed at 37 deg.C with 5% CO2Culturing for 48 h. After 48h, cells were spun into growth medium and then counted in a cell counter, the cell suspension was diluted to the desired density, and 50 μ l of cell suspension was taken to a 96-well plate with 25000 cells per well. 37 ℃ and 5% CO2Culturing for 12h, and completely attaching the cells to a 96-well plate after culturing for 12 h.
3) Adding medicine and incubating: according to the experimental design, 10 mul of the prepared compound solution to be tested with the prepared concentration is added into each hole of a 96-hole plate, the temperature is 37 ℃, and the CO content is 5 percent2Incubate for 1 h.
4) Adding a false virus: 40 μ L of virus supernatant was added to each well of a 96-well plate. 37 ℃ and 5% CO2And culturing for 24 h. After 24h, the medium was replaced with fresh medium at 100. mu.L/well. 37 ℃ and 5% CO2And culturing for 24 h.
5) Measurement: the well plate was equilibrated to room temperature before measurement, and 30uL of renin luciferase reagent was added to each well; mixing on a shaker for 2 minutes to induce cell lysis; incubate for 5 minutes at room temperature to stabilize the luminescent signal; fluorescence data on the microplate reader was recorded.
Relative fluorescence intensity (%) between control and dosed wells treated with DMSO was calculated using the following equation:
relative fluorescence intensity (%). well fluorescence/control well fluorescence 100%
The inhibition rate of the compound on the COVID-19S protein pseudovirus infected cells is 100 percent to relative fluorescence intensity (%).
TABLE 4 experimental groups for inhibiting infection of COVID-19S protein virus and concentrations tested
TABLE 5 results of inhibiting COVID-19S protein virus infection
According to the above experimental method, the results of activity detection of the selected compounds are shown in table 5 and fig. 4A to 4C, the candidate compounds have good inhibitory effect on the infection of the COVID-19S protein pseudovirus on 293T cells expressing hACE2, and the inhibitory rate on pseudovirus infection can reach more than 75% when the concentration of the compound is more than 250 μ M.
In conclusion, the method for targeting and blocking the active molecule of the COVID-19S protein based on virtual screening can quickly and efficiently screen active lead compounds from a large number of compounds, and provides theoretical basis and practical guidance for further structure optimization of the compounds.
The above description of exemplary embodiments has been presented only to illustrate the technical solution of the invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those skilled in the art. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to thereby enable others skilled in the art to understand, implement and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.
Sequence listing
<110> university of Large Community
<120> method for screening small molecule inhibitors of COVID-19 spinous process proteins, active molecules screened thereby, and methods for screening small molecule inhibitors of COVID-19 spinous process proteins
Use of
<130> 2020
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 804
<212> PRT
<213> Homo sapiens
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Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Pro His Asp Glu Thr Tyr
485 490 495
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
500 505 510
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
515 520 525
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
530 535 540
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
545 550 555 560
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
565 570 575
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
580 585 590
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
595 600 605
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
610 615 620
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
625 630 635 640
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
645 650 655
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
660 665 670
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
675 680 685
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
690 695 700
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
705 710 715 720
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
725 730 735
Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val Ile
740 745 750
Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg Lys
755 760 765
Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile Asp
770 775 780
Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp Val
785 790 795 800
Gln Thr Ser Phe