WO2021151264A1 - 酰基化螺旋霉素在制备治疗冠状病毒感染疾病药物上的应用 - Google Patents
酰基化螺旋霉素在制备治疗冠状病毒感染疾病药物上的应用 Download PDFInfo
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- WO2021151264A1 WO2021151264A1 PCT/CN2020/081204 CN2020081204W WO2021151264A1 WO 2021151264 A1 WO2021151264 A1 WO 2021151264A1 CN 2020081204 W CN2020081204 W CN 2020081204W WO 2021151264 A1 WO2021151264 A1 WO 2021151264A1
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- isovalerylspiramycin
- coronavirus
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
Definitions
- the present invention relates to new uses of antibiotics, especially macrolide antibiotics or isovalerylspiramycin compounds or combinations thereof. Specifically, the present invention relates to acylated isovalerylspiramycin analogs or their combinations in the preparation of The application of drugs for the treatment of coronavirus infections.
- the isovalerylspiramycin analogue was produced by Professor Wang Yiguang's research group from the Bioengineering Department of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences.
- the carbamycin 4"-isovaleryltransferase gene was integrated into the spiramycin-producing bacteria by using homologous recombination technology.
- Streptomyces spiramyceticus F21 A series of antibiotics produced by genetically engineered bacteria constructed on the chromosome; as a major project of the Ministry of Science and Technology, this hybrid antibiotic is a national first-class new drug with the generic name colimycin; it is the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences A national category 1 innovative drug jointly developed with Shenyang Tonglian Group Co., Ltd.
- Climycin is a multi-component small molecular structure: including isovalerylspiramycin III, isovalerylspiramycin II, isovalerylspiramycin A mixed chemical compound composed of three main components of element I and a certain amount of butyryl, propionyl, acetylspiramycin III, butyryl, propionyl, and acetylspiramycin II.
- Isovalerylspiramycin II Molecular formula: C50H84N2O16 Molecular weight: 968.00
- Isovalerylspiramycin III Molecular formula: C51H86N2O16 Molecular weight: 982.00
- the main component structure is as follows:
- the I, II, and III in the above formula are the isovalerylspiramycin compounds I, II, and III.
- the mechanism of action of spiramycin is to specifically bind to the 50S subunit of the bacterial ribosome and interfere with bacterial protein synthesis by preventing the extension of the new peptide chain.
- the degradation product of spiramycin in the body is that the activity of the degradation product after decarburose is reduced or disappears, and after 4" acylation, it becomes the degradation pathway of defoloxose, forming Plattella with antibacterial activity.
- A1 and other products. 4" isovalerylated side chain can also improve its stability in vivo, delay hydrolysis, and prolong the pharmacokinetic characteristics of half-life. Studies have shown that when spiramycin is acylated at the 4" position, its antibacterial activity is enhanced in vivo, including bacteria resistant to certain macrolide antibiotics.
- isovalerylspiramycin analogs have strong antibacterial activity, and also have significant inhibitory activity against mycoplasma and chlamydia, and have no obvious cross-resistance with similar drugs.
- drug-resistant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and drug-resistant Streptococcus pyogenes
- it also has a good effect on some ⁇ -lactamase-producing bacteria.
- Gram-negative bacteria such as Clostridium difficile, Influenza bacillus
- fungus Candida albicans also have better activity.
- the isovalerylspiramycin analog has the advantages of low toxicity, small dose, less frequency of medication and convenient taking, it is the first choice for patients to pursue safe and effective drugs within the scope of indications; the development of its potential new uses is also significant Meaning.
- the virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an envelope, with a diameter of about 80-120nm. Its genetic material is the largest among all RNA viruses, and it only infects humans, mice, pigs, cats, dogs, and poultry. vertebrate.
- coronavirus family In 1975, the National Virus Nomenclature Committee officially named the coronavirus family. So far, about 15 different coronavirus strains have been discovered, which can infect a variety of mammals and birds, and some can cause illness in humans.
- coronavirus is the seventh member of the coronavirus family that can infect humans.
- 2019-nCoV is the third type of coronavirus that has appeared in humans in the past 20 years.
- SARS infectious atypical pneumonia
- MERS Middle East respiratory syndrome
- SARS infectious atypical pneumonia
- MERS Middle East respiratory syndrome
- the first object of the present invention is to provide an acylated spiramycin compound or its composition in the preparation of drugs for the treatment of coronavirus infections; in particular, isovalerylspiramycin compound or composition used in coronavirus
- the application of infectious disease drugs specifically, the application of climycin in the treatment of coronavirus infection drugs.
- the second object of the present invention is to provide the use of acylated spiramycin compounds and their compositions in the preparation of drugs for the treatment of 2019-nCoV coronavirus infections; in particular, isovaleryl spiramycin compounds and their compositions are used in Application of drugs for the treatment of 2019-nCoV coronavirus infection disease. Specifically, the application of colimycin in the treatment of diseases caused by the 2019-nCoV virus.
- the third object of the present invention is to provide the use of acylated spiramycin compounds or their compositions in the preparation of drugs for the treatment of complications caused by coronavirus infection; in particular, isovalerylspiramycin compounds or compositions are used in the treatment of The application of complication drugs caused by coronavirus infections; specifically, the application of climycin in the treatment of complication drugs caused by coronavirus diseases. Specifically, the application of colimycin in the treatment of complications caused by 2019-nCoV pneumonia.
- acylated spiramycin compounds or their compositions in the preparation of drugs for the treatment of coronavirus infections; in particular, the use of isovalerylspiramycin compounds or their combinations in drugs for coronavirus infections; specifically, Application of Celimycin in the treatment of coronavirus infections.
- acylated spiramycin compounds and their compositions in the preparation of drugs for the treatment of coronavirus infections; in particular, applications of isovalerylspiramycin compounds and their compositions in the treatment of 2019-nCoV disease drugs. Specifically, the application of colimycin in drugs for the treatment of 2019-nCoV disease.
- antibiotics especially macrolide antibiotic compounds
- drugs for the treatment of diseases such as coronavirus infections
- drugs for 2019-nCoV infections especially the application of drugs for 2019-nCoV infections.
- the acylated acylspiramycin compounds in the present invention are at least selected from the group consisting of isovalerylspiramycin I, isovalerylspiramycin II, and isovalerylspiramycin obtained from the preparation of cleritromycin by genetic engineering methods III One of the three main components, and a certain amount of butyryl, propionyl, acetylspiramycin III, butyryl, propionyl, acetylspiramycin II and other 9 components or a combination thereof.
- It is preferably a compound selected from isovalerylspiramycin, more preferably one of isovalerylspiramycin I, II, and III, its structural analogues or a combination of at least two of them, or a cleremycin, wherein Mycin is a new national class of drugs, which of course includes any of the above-mentioned compounds or their combinations obtained by chemical or biological methods, or the analogs of climycin.
- the coronaviruses of the present invention include alpha and beta coronaviruses, preferably: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and 2019-nCoV, preferably SARS- CoV, MERS-CoV, and 2019-nCoV, where SARS is severe acute respiratory syndrome, MERS is Middle East respiratory syndrome, and 2019-nCoV (COVID-19) new coronavirus infection.
- the coronavirus infection diseases of the present invention include respiratory tract, digestive tract and nervous system diseases, including but not limited to cold, fever, frontal sinusitis, otitis media, pharyngitis, chronic bronchitis, pneumonia, pleural effusion, various respiratory syndromes Symptoms, acute gastroenteritis, cardiopulmonary disease, low immunity, repeated infection, lung injury, organ failure and other diseases.
- various diseases and complications caused by SARS, MERS and 2019-nCoV including various infectious diseases.
- acylated spiramycin compound or its composition of the present invention binds to the Mpro inhibitory site of the major protease of coronavirus.
- the acylated spiramycin compound or its composition of the present invention binds to the coronavirus S protein and its host cell receptor ACE2 protein.
- the isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III, or clidinomycin of the present invention binds to the coronavirus S protein and its host cell receptor ACE2 protein.
- isovalerylspiramycin I isovalerylspiramycin II
- isovalerylspiramycin III or cleritromycin of the present invention binds to the Mpro inhibitory site of the major protease of coronavirus.
- the therapeutic drug of the present invention includes one of the acylated spiramycin compounds of the present invention, or a combination of at least two of them, or climycin as the first active ingredient of the drug, supplemented by the second active ingredient of the drug, so
- the second active ingredient of the drug can be selected from antiviral drugs and/or anti-AIDS drugs, and the first active ingredient and the second active ingredient of the drug can be independent preparations, or they can be compounded into one preparation.
- the second active ingredient of the drug can be a protease inhibitor, a fusion protein inhibitor, a nucleoside reverse transcriptase inhibitor, an immunosuppressive agent, etc., and is selected from at least one of the following active ingredients: indinavir (Indinavir ), Saquinavir, Lopinavir, Carfilzomib, Ritonavir, Remdesivir or Remdesivir RDV, GS-5734 ), atazanavir, darunavir, tiranavir, fosamprenavir, szatovir, pretovir, ribavirin, abacavir, bortezomib, etegavir , Montelukast, deoxyrheum glycosides, polydatin, mountain bean root chalcone, disulfiram, carmofur, shikonin, ebselen, Tideglusib, PX-12, TDZD8, cinnamon thiamine, cyclamate Drugs or
- Figure 1 is one of the schematic diagrams of the binding of isovalerylspiramycin I and angiotensin-converting enzyme ACE2 of the present invention
- Figure 2 is the second schematic diagram of the combination of isovalerylspiramycin I and angiotensin-converting enzyme ACE2 of the present invention
- FIG. 3 is a schematic diagram of the binding of isovalerylspiramycin I and spike protein of the present invention
- Figure 4 is a schematic diagram of the 3D structure of the SARS-CoV main protease (Mpro, also known as 3CL protease) and its inhibitor binding site, that is, a schematic diagram of its role in mediating viral replication and transcription functions;
- Mpro SARS-CoV main protease
- 3CL protease SARS-CoV main protease
- FIG. 5 is a schematic diagram of the binding of isovalerylspiramycin I and 3CL of the present invention.
- FIG. 6 shows the docking of several different drugs with SARS-CoV Mpro
- FIG. 7 Schematic diagram of the inhibition of CCR5 expression by climamycin
- FIG 8 shows the flexibility analysis of isovalerylspiramycin I (CM926) and SARS-S system
- Figure 9 is a schematic diagram of the binding of isovalerylspiramycin I (CM926) to SARS-S; (A) 2D binding mode of SARS-S and CM926; (B) binding model of CM926 on the surface of SARS-S molecules; (C) The 3D combination mode of SARS-S and CM926;
- FIG 10 shows the flexibility analysis of isovalerylspiramycin I (CM926) and SARS-M system
- Figure 11 is a schematic diagram of the binding of isovalerylspiramycin I (CM926) to SARS-M; (A) 2D binding mode of SARS-M and CM926; (B) binding model of CM926 on the surface of SARS-M molecule; (C) ) The 3D combination mode of SARS-M and CM926;
- Figure 12 shows the flexibility analysis of isovalerylspiramycin I (CM926) and ACE2 system
- Figure 13 shows the binding of isovalerylspiramycin I (CM926) and ACE2
- A 2D binding mode of ACE2 and CM926
- B binding model of CM926 on the surface of ACE2 molecule
- C 3D binding of ACE2 and CM926 model
- Figure 14 shows the flexibility analysis of isovalerylspiramycin I (CM926) and ACE2-SARS system
- Figure 15 is a schematic diagram showing the binding of isovalerylspiramycin I (CM926) to ACE2-SARS, (A) the 2D binding mode of ACE2-SARS and CM926; (B) the binding model of CM926 on the surface of ACE2-SARS molecules; (C) The 3D combination mode of ACE2-SARS and CM926;
- Figure 19 One of the improvement of lung inflammation before and after treatment with Climycin
- Figure 21 shows the improvement of lung inflammation in the patient of case 1 before and after treatment with climycin in a specific case.
- Isovalerylspiramycin I is one of the main active substances of cleritromycin and one of the characteristic components of cleritromycin; molecular docking shows that isovalerylspiramycin I can interact with spike -ACE2 protein complex binds at multiple sites.
- Isovalerylspiramycin I and ACE2 have a strong binding, and the binding position is located in the active site pocket of ACE2 ( Figure 1). This structure is related to the catalytic function of ACE2. When ACE2 performs its catalytic function, the three-dimensional structure of this region needs to be changed. The binding of isovalerylspiramycin I will limit the changes of the three-dimensional structure of ACE2. Therefore, isovalerylspiramycin I has ACE2 inhibition. effect.
- the isovalerylspiramycin compounds II and III also bind to spike protein, and the binding position is located in the active site pocket of ACE2 ( Figure omitted).
- the S protein (spike) of the coronavirus is combined into a trimer, which contains about 1300 amino acids, and belongs to the first class of membrane fusion protein (Class I viral fusion protein). Similar viral membrane fusion proteins also include the Env protein of HIV and influenza. The HA protein of Ebola virus and the Gp protein of Ebola virus. The S protein determines the host range and specificity of the virus, and is also an important site for host neutralizing antibodies.
- Figures 3a and 3b show that isovalerylspiramycin I also binds near the RBD domain of the spike protein. Therefore, isovalerylspiramycin I may inhibit the binding of SARS virus to the host receptor.
- valerylspiramycin I of the present invention can inhibit SARS virus or influenza virus.
- the isovalerylspiramycin compounds II and III also bind to the spike protein, so the isovalerylspiramycin compounds II, III, or climycin can inhibit SARS virus or influenza virus.
- Betacoronavirus is a protein-encapsulated single-stranded positive-stranded RNA virus that parasites and infects higher animals (including humans). In the position of the evolutionary tree, it is adjacent to the SARS (causing "SARS" in 2002) virus and the SARS-like virus group, but does not belong to the SARS and SARS-like virus group; the evolutionary common outer group is A HKU9-1 coronavirus parasitizing fruit bats.
- the author of the article used the calculation method of molecular structure simulation to conduct a structural docking study on the S-protein of the coronavirus SARS-CoV-2 and the human ACE2 protein, and obtained surprising results. Although four of the five key amino acids that bind to the ACE2 protein in the S-protein of the coronavirus SARS-CoV-2 have changed, the changed amino acids have perfectly maintained the SARS virus S-protein and the ACE2 protein as a whole. The original structural conformation of ACE2 protein interaction.
- the new structure of the new coronavirus SARS-CoV-2 interacts with the ACE2 protein due to a small number of missing hydrogen bonds (compared to the decrease in the effect of the SARS virus S-protein and ACE2), it still reaches a very strong level. Free energy of binding (-50.6kcal/mol). This result indicates that the new coronavirus SARS-CoV-2 infects human respiratory epithelial cells through the molecular mechanism of S-protein and human ACE2 interaction.
- the S-protein structure of SARS and the S-protein structure of 2019-nCoV virus have almost the same 3D structure of the RBD domain, which directly interacts with the host cell receptor ACE2.
- 2019-nCoV virus S-protein Based on the S-protein structure of SARS, the structure of 2019-nCoV virus was simulated, and then based on the structure of the composite crystal composed of SARS and ACE2, the ability of 2019-nCoV virus S-protein to interact with human ACE2 molecules was evaluated.
- the binding free energy between 2019-nCoV virus S-protein and human ACE2 is -50.6kcal/mol, which is 28kcal/mol higher than SARS's -78.6kcal/mol.
- the binding of 2019-nCoV virus S-protein to human ACE2 is relatively weak, but this affinity is still considered strong.
- the combination of isovalerylspiramycin I and ACE2 protein will limit the three-dimensional structure changes of ACE2, indicating that isovalerylspiramycin I has potential ACE2 inhibitory activity.
- SARS infection will lead to a decrease in ACE2 expression, which in turn leads to increased lung damage; based on the combination of small molecules and proteins, which can enhance protein stability, the combination of isovalerylspiramycin I and ACE2 can inhibit the function of ACE2 on the one hand, and on the other On the one hand, it can increase the stability of ACE2, and it is possible to inhibit the decrease of ACE2 expression after SARS infection.
- ACE2 is expressed in human cornea and conjunctiva.
- Isovalerylspiramycin I may have an inhibitory effect on both the coronavirus S protein and its host cell receptor ACE2, so it may have an inhibitory effect on coronavirus infection.
- FIG 4 shows that the SARS-CoV main protease (Mpro), also known as the 3CL protease, plays a key role in mediating viral replication and transcription functions. Between domain I and domain II is the binding site of the inhibitor.
- Mpro SARS-CoV main protease
- Figure 5 shows that the binding sites of isovalerylspiramycin I and 3CL are consistent with the binding sites of 3CL inhibitors.
- Mpro(3CL) has been used by the Shanghai Institute of Medicine, Chinese Academy of Sciences as a screening model for molecular simulation technology to screen coronavirus drugs. Relevant screening results will be released on 2020.1.25. Therefore, isovalerylspiramycin I has great potential value in anti-viral infection.
- isovalerylspiramycin III Similar to its structure isovalerylspiramycin III, isovalerylspiramycin II, and butyryl, propionyl, acetylspiramycin III, butyryl, propionyl, acetylspiramycin II and other climycin
- the active ingredient and climycin have great potential value in anti-viral infection, especially for 2019-nCoV virus infection.
- the present invention also compares the docking of several different drugs with SARS-CoV Mpro, see Figure 6 and Table 1. It can be seen that among these drugs, the docking of isovalerylspiramycin I and SARS-CoV Mpro is significantly stronger than other drugs, not only superior to similar drugs, but also superior to lopinavir and ritola with antiviral effects. Nawei. Other commonly used antibiotics in clinical practice are not only weaker than isovalerylspiramycin I, and cannot completely bind to the inhibitory site, which makes it difficult to effectively inhibit Mpro.
- Isovalerylspiramycin I can perfectly bind to the Mpro inhibitory site due to the presence of its isovaleryl group, indicating that isovalerylspiramycin I, isovalerylspiramycin III, and isovalerylspiramycin II, and butyryl, propionyl, acetylspiramycin III, butyryl, propionyl, acetylspiramycin II and other active ingredients of colimycin and colimycin in coronavirus infection, inflammation and secondary Infection and other aspects have good clinical value.
- Table 1 The binding energy of several different drugs with SARS-CoV M pro
- antibiotics such as isovalerylspiramycin I, spiramycin I, azithromycin, erythromycin, moxifloxacin, clarithromycin, and macrolide antibiotics have certain effects. It can be a potential drug for the treatment of new coronavirus infections.
- the main symptoms of new viral infections reported are immune injury response leading to interstitial pneumonia, multiple organ function damage, lung damage, etc.
- the present invention adopts isovalerylspiramycin I and proteins: SARS-Spike, SARS-Mpro, ACE2, and ACE2-SARS-Spike respectively to do the docking, and then perform molecular dynamics simulation.
- Iovalerylspiramycin I small molecule CM9266 is energy-optimized in MOE to obtain a low-energy three-dimensional conformation as a ligand.
- the binding site is defined as follows: SARS-S (Cys323, Ser358), SARS-M (Glu166, Phe140, Cys145), ACE2 (Lys74, Ser106, Gln102, Asp350), ACE2-SARS (Asp38, Gln42, Gln325, Glu329, Arg426, Tyr436).
- AMBER10 EHT force field and R-field implicit solvent model.
- the docking process adopts a flexible induced fit mode, the side chain of the amino acid binding pocket can be optimized and adjusted according to the ligand conformation, and the weight of restraining side chain rotation is set to 10.
- the binding mode of the ligand is first sorted by the London dG scoring function, and the first 30 conformations are further optimized and the GBVI/WSA dG method is used to evaluate the binding free energy again. Select the binding mode with the best score (the lowest binding free energy) and perform molecular dynamics simulation.
- the RMSD value of the protein SARS-S skeleton atoms is less than 2.5 angstroms, while the RMSD value of isovalerylspiramycin I (small molecule CM926) is less than 5.0 angstroms.
- the entire system has reached equilibrium within the simulation time. The force field used in system simulation is appropriate.
- Cluster analysis is performed on the trajectories obtained by kinetic simulation, and the cluster center of the trajectory after the system is stabilized is taken as the final binding mode conformation of isovalerylspiramycin I and protein SARS-S, as shown in FIG. 9.
- Isovalerylspiramycin I is located around the SARS-S binding site. However, there is no obvious hydrogen bond and Pi-Pi stacking interaction between isovalerylspiramycin I and SARS-S. Most of the isovalerylspiramycin I is exposed to the solvent. Van der Waals interactions are formed between CM926 and residues around the binding site.
- the RMSD value of the protein SARS-M skeleton atoms is less than 2.5 angstroms, while the RMSD value of the small molecule CM926 is less than 3.0 angstroms.
- the entire system has reached equilibrium within the simulation time, indicating that the force field used in the system simulation is appropriate of.
- Cluster analysis is performed on the trajectories obtained by the kinetic simulation, and the cluster center of the trajectory after the system is stabilized is taken as the final binding mode conformation of the small molecule CM926 and the protein SARS-M, as shown in Figure 11.
- the binding sites of CM926 and SARS-M formed proper spatial complementarity.
- a hydrogen bond and electrostatic interaction are formed between CM926 and SARS-M.
- the nitrogen atom in the amino group of CM926 is regarded as a hydrogen bond donor, forming hydrogen bonds with the side chain oxygen atoms of Glu166 and Gln189, respectively.
- the positively charged nitrogen atom in the amino group forms an electrostatic interaction with the negatively charged side chain oxygen atom of Glu166.
- VDW interactions are formed between CM926 and surrounding residues. These main interactions promote the binding between CM926 and SARS-M protein.
- the RMSD value of the protein ACE2 backbone atoms is less than 3.0 angstroms, while the RMSD value of the small molecule CM926 is less than 3.0 angstroms.
- the entire system has reached equilibrium within the simulation time, indicating that the force field used in the system simulation is appropriate.
- Cluster analysis was performed on the trajectories obtained by kinetic simulation, and the cluster center of the trajectory after the system was stabilized was taken as the final binding mode conformation of small molecule CM926 and protein ACE2, as shown in Figure 13.
- the binding sites of CM926 and ACE2 formed proper spatial complementarity.
- a hydrogen bond and electrostatic interaction are formed between CM926 and ACE2.
- the oxygen atom in the hydroxyl group of CM926 is regarded as a hydrogen bond donor and forms a hydrogen bond with the side chain oxygen atom of Asp206.
- the nitrogen atom in the CM92 amino group is regarded as a hydrogen bond donor, forming hydrogen bonds with the side chain oxygen atoms of Asp206 and Asp350, respectively.
- the oxygen atom in the carbonyl group of CM926 is regarded as a hydrogen bond acceptor and forms a hydrogen bond with the side chain nitrogen atom of Lys562.
- the RMSD value of the protein ACE2-SARS skeleton atoms is less than 5.0 angstroms, and the RMSD value of the small molecule CM926 is less than 3.0 angstroms.
- the entire system has reached equilibrium within the simulation time, indicating that the force field used in the system simulation is appropriate of.
- Cluster analysis is performed on the trajectories obtained by kinetic simulation, and the cluster center of the trajectory after the system is stabilized is taken as the final binding mode conformation of small molecule CM926 and protein ACE2-SARS, as shown in Figure 15.
- the binding sites of CM926 and ACE2-SARS formed proper spatial complementarity.
- a hydrogen bond and electrostatic interaction are formed between CM926 and ACE2-SARS.
- the nitrogen atom in the amino group of CM926A is regarded as a hydrogen bond donor and forms a hydrogen bond with the side chain oxygen atom of Glu35 of the ACE2 subunit in the complex protein.
- the positively charged nitrogen atom in the amino group forms an electrostatic interaction with the negatively charged side chain oxygen atom of Glu35.
- VDW interactions are formed between CM926 and surrounding residues. These main interactions promote the binding between CM926 and the protein ACE2-SARS.
- the binding free energy ⁇ Gtotal of CM926 and protein is shown in Table 1.
- the binding free energy is composed of 4 parts.
- the van der Waals interaction energy term is represented by ⁇ Evdw
- the electrostatic interaction energy term is represented by ⁇ Eele
- the polar interaction energy term of the solvent is represented by ⁇ Gpolar
- the non-polar interaction energy term of the solvent is represented by ⁇ Gnonpolar.
- the binding free energy of small molecule CM926 and proteins SARS-S, SARS-M, ACE2 and ACE2-SARS are: -3.26, -6.20, -9.16 and -6.76kcal/mol, respectively.
- the predicted approximate IC50 value is calculated by the following formula:
- R 8.314 and T is 300K
- the final predicted approximate IC50 values are 1817.33 ⁇ M, 30.27 ⁇ M, 0.21 ⁇ M and 11.82 ⁇ M respectively.
- Test product Climycin, provided by Shenyang Tonglian Group Co., Ltd. DMSO is made into a mother liquor of 20mg/ml and stored in a refrigerator at 4°C. In the experiment, the culture solution was diluted to an appropriate concentration and then the experiment was carried out. Store in a refrigerator at 4°C.
- Ribavirin injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., batch number 31712252, specification 100mg/ml, diluted to the required concentration when used, and stored in a refrigerator at 4°C.
- Azithromycin commercially available
- HCT-8 cells human liver cancer cells Huh7.5 cells and human diploid cells MRC-5 cells are all passaged and preserved in our laboratory (Institute of Livelihoods, Chinese Academy of Sciences), and are stored in 10% fetal bovine serum (inactivated fetal bovine). Serum) and 1% double antibodies (penicillin and streptomycin) in DMEM or 1640 medium, 37°C, 5% CO2 incubator. Passage once every 2-3 days.
- HCoV-229E was passaged in Huh7.5 cells and stored in a refrigerator at -80°C.
- HCoV-OC43 was passaged in HCT-8 cells and stored in a refrigerator at -80°C.
- HCT-8 cells as an example: add 3ml of 0.25% Trypsin-EDTA to a culture flask full of HCT-8 cells, digest for 1-2 minutes at 37°C, discard the digestion solution, add the culture solution and pipette, 1 : Passage 4, passage once every 2-3 days.
- 200,000 cells per milliliter Inoculate a 96-well cell culture plate, 0.1ml per well, 37°C, 5% CO2 and incubate overnight. After the cells grow into a monolayer conduct experiment.
- A drug concentration with cumulative inhibition rate ⁇ 50%
- B inhibition rate with cumulative inhibition rate> 50%
- C inhibition rate with cumulative inhibition rate ⁇ 50%
- D log dilution factor
- the infection was administered at the same time: the CPE method determined that the IC50 of the HCoV-229E strain after 0h administration of Climycin was 3.14 ⁇ 0.80 ⁇ g/ml, and the selection index SI was 13.0; RBV was administered at 0h to HCoV The IC50 of -229E is 7.06 ⁇ 0.91 ⁇ g/ml, and the selection index SI is> 14.2; the IC50 of azithromycin to HCoV-229E is 16.78 ⁇ 3.48 ⁇ g/ml, and the selection index SI is 5.0.
- Dosing 2h after infection The IC50 of CPE method for the HCoV-229E strain after 2h dosing is 2.36 ⁇ 0.30 ⁇ g/ml, the selection index SI is 15.1; the IC50 of RBV for HCoV-229E after 2h dosing is 2.36 ⁇ 0.30 ⁇ g/ml 6.24 ⁇ 2.07 ⁇ g/ml, the selection index SI is >16.0; the IC50 of azithromycin to HCoV-229E after 2h administration is 9.55 ⁇ 6.75 ⁇ g/ml, and the selection index SI is 6.7.
- the IC50 of the HCoV-OC43 strain determined by the CPE method at 0h was 25.87 ⁇ g/ml, and the selection index SI was 2.2; RBV was administered at 0h to HCoV
- the IC50 of -OC43 is 11.11 ⁇ g/ml, and the selection index SI is >9.0; Azithromycin has no inhibitory effect on HCoV-OC43 when administered at 0h.
- the IC50 of the HCoV-OC43 strain of the HCoV-OC43 strain determined by the CPE method at 0h was 5.93 ⁇ g/ml, and the selection index SI was 3.2; RBV was administered at 0h to HCoV
- the IC50 of OC43 is 63.75 ⁇ g/ml, and the selection index SI is >1.6; Azithromycin has no inhibitory effect on HCoV-OC43 when administered at 0h.
- Table 4 The inhibitory effect of drugs on HCoV-OC43 in HCT-8 cells (IC 50 ) (CPE method)
- Table 5 The inhibitory effect of drugs on HCoV-OC43 in MRC-8 cells (IC 50 ) (CPE method)
- Figure 16 shows that the inhibitory effect of climycin on the CPE induced by the HCoV-229E strain in Huh 7.5 cells can be seen
- the IC50 of the HCoV-229E strain of HCoV-229E strain determined by the CPE method at 0h was 3.14 ⁇ 0.80 ⁇ g/ml, the selection index SI was 13.0; RBV was given at 0h
- the IC50 of the drug to HCoV-229E is 7.06 ⁇ 0.91 ⁇ g/ml, and the selection index SI is> 14.2; the IC50 of azithromycin to HCoV-229E is 16.78 ⁇ 3.48 ⁇ g/ml, and the selection index SI is 5.0.
- the IC50 for HCoV-229E strain after 2h administration of CPE by CPE method was 2.36 ⁇ 0.30 ⁇ g/ml, and the selection index SI was 15.1; the IC50 for HCoV-229E after 2h administration of RBV was 6.24 ⁇ 2.07 ⁇ g/ml , The selection index SI was> 16.0; the IC50 of azithromycin to HCoV-229E was 9.55 ⁇ 6.75 ⁇ g/ml after 2h administration, and the selection index SI was 6.7.
- the CPE method determined that the IC50 of Climycin against HCoV-OC43 strain at 0h was 25.87 ⁇ g/ml, and the selection index SI was 2.2; the IC50 of RBV against HCoV-OC43 at 0h was 11.11 ⁇ g/ml, the selection index SI is >9.0; azithromycin administration at 0h has no inhibitory effect on HCoV-OC43.
- the IC50 of the HCoV-OC43 strain measured by CPE method at 0h was 5.93 ⁇ g/ml, and the selection index SI was 3.2; the IC50 of RBV at 0h was 63.75 for HCoV-OC43. ⁇ g/ml, the selection index SI is >1.6; Azithromycin administration at 0h has no inhibitory effect on HCoV-OC43.
- colimycin has an inhibitory effect on both HCoV-OC43 and HCoV-229E strains; RBV has an inhibitory effect on both HCoV-OC43 and HCoV-229E strains; azithromycin has an inhibitory effect on HCoV-229E.
- the strain has an inhibitory effect, but has no inhibitory effect on the HCoV-OC43 strain.
- colimycin has clear in vitro anti-coronavirus HCoV-229E and HCoV-OC43 activity, and is better than azithromycin.
- the anti-coronavirus HCoV-229E and HCoV-OC43 activity of RBV is comparable to the previous results in the literature and this experiment, indicating that the experimental system is established.
- the present invention adopts the above method to detect isovalerylspiramycin I, isovalerylspiramycin II, and isovalerylspiramycin III of the present invention, and the results of isovalerylspiramycin I are higher than that of colimycin. , Isovalerylspiramycin II and isovalerylspiramycin III are close to colimycin.
- the present invention also uses the above-mentioned method to carry out the inhibition of colimycin, isovalerylspiramycin I, isovalerylspiramycin II, and isovalerylspiramycin III to inhibit Middle East Respiratory Syndrome Coronavirus (MERS-CoV), severe Acute respiratory syndrome coronavirus SARS-CoV, human coronavirus HKU1, human coronavirus NL63 (HCoV-NL63) in vitro experiments, the results are higher than the drug ribavirin or azithromycin.
- MERS-CoV Middle East Respiratory Syndrome Coronavirus
- SARS-CoV severe Acute respiratory syndrome coronavirus
- HKU1 human coronavirus NL63
- the present invention conducted in vitro experiments on the anti-2019-nCoV activity of cleritromycin, isovalerylspiramycin I, isovalerylspiramycin II, and isovalerylspiramycin III, as follows:
- Test drugs Climycin, isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III.
- VeroE6 cells preserved by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.
- Virus 2109-nCoV, with a titer of 105TCID50/ml, stored at -80°C by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.
- the virus titer used is 100TCID50.
- Test drug Climycin was tested at two concentrations: 10 ⁇ g/ml and 2 ⁇ g/ml.
- a sterile 96-well culture plate add 200 ⁇ l of Vero E6 cells at a concentration of 5 ⁇ 104cell/ml to each well, and incubate at 37°C with 5% CO2 for 24 hours;
- test drug was diluted to 2 concentrations, each with 5 replicate holes, 100 ⁇ l per hole, and then an equal volume of 100 TCID50 virus was added to each hole for 1 hour;
- CPE cytopathic
- the drugs were set at two concentrations, which can effectively inhibit the replication of 2019-nCoV on the cells.
- the results are shown in Table 7.
- colinomycin can inhibit the replication of 2109-nCoV in cells at a concentration of 2 ⁇ g/ml, suggesting that colinomycin has in vitro anti-2109-nCoV activity.
- isovalerylspiramycin I isovalerylspiramycin II
- isovalerylspiramycin III also have in vitro anti-2109-nCoV activity.
- Calinomycin significantly improves the pathological changes of inflammation and edema in important organs of the whole body, especially the lung, liver, kidney and heart, brain, stomach, intestines and other organs. It is antibacterial and improves inflammation and edema in immunodeficiency nude mice. By activating the immune mechanism in the body, it has the effect of antibacterial and improvement of tissue damage.
- PI3K/Akt/mTOR pathway can be inhibited; it can be used in animals
- Climycin and isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III anti-inflammatory and immunomodulation-inhibition of PI3K/Akt/mTOR pathway, thereby reducing IL-6 And IL-8 levels indicate.
- Climycin can inhibit the expression of mTOR and p-mTOR by treating A549 cells for 48 hours. Climbing can inhibit the expression of P70S6K1 (ribosomal protein S6 kinase) and p-P70S6K1 downstream of mTOR. Studies have shown that after mTOR is inhibited, IL-6 and IL-8 levels will be significantly reduced.
- the new coronavirus (2019-nCoV), like other coronaviruses, stimulates the patient's innate immune system, causing the body to release a large amount of cytokines, causing a cytokine storm and acute inflammation. This can lead to more fragile blood vessels throughout the body, leading to acute respiratory distress and multiple organ failure.
- the climycin of the present invention has an antiviral effect and can be used in the early stage to reduce the effect of the virus on the immune system. For severely ill patients, by controlling the cytokine storm and acute inflammatory response, see Figure 18, the colimycin can significantly reduce the lung And other organ damage.
- Figure 18 is a schematic diagram of the repair of lung injury caused by climycin.
- the administration of colimycin for 6 days can improve the tissue inflammation and inflammation caused by infection in mice. Tissue cell damage; HE pathological staining shows that it is more effective in improving body tissue inflammation and damage; it indicates that cleritromycin may improve the body's immune system function, repair tissue damage, and promote recovery;
- Calinomycin also has the characteristics of high safety, wide tissue distribution, high tissue concentration, long half-life, and obvious antipyretic effect. It also has anti-inflammatory, immune-regulating, and anti-edema effects. It is resistant to drug-resistant Klebsiella pneumoniae and Acinetobacter baumannii has good therapeutic effects and has potential clinical application value in the battle against coronavirus infection. Combining the above molecular simulation calculation results, it can be considered that clinomycin has good clinical value in terms of coronavirus infection, inflammatory response, and secondary infection.
- Light type Climycin tablets, 0.4g each time, once a day, orally after a meal, for 7 consecutive days, and enter the follow-up observation period for 30 days after the treatment.
- Figure 19 shows the improvement of lung inflammation in one of the patients before and after treatment with clinomycin. It can be seen from the figure that the lung inflammation was significantly improved after 5 and 10 days of treatment with clarithromycin;
- Figure 20 is another patient, (A) is suffering CT scan on the first day of illness, (B) is the CT scan on the fifth day of illness, (C) is the CT scan on the 6th day of illness (the day when climycin treatment is started), (D) is the day of illness CT scan on 8 days; (E) is the CT scan on the 11th day of illness.
- colimycin can quickly and effectively eliminate the new coronavirus, significantly improve clinical symptoms and lung inflammation, improve the prognosis of patients with new coronary pneumonia, and shorten hospitalization time. Climycin is convenient for clinical application, and it is orally administered once a day without obvious adverse reactions.
- RT-PCR of throat swab showed positive for new coronavirus, oxygen saturation was 83% under oxygen inhalation, oxygen partial pressure in blood gas analysis was 64mmHg, blood routine examination was normal, liver and kidney function was normal, and history of hypertension was always taken. Suppress the drug, and the blood pressure is stable at ordinary times. After admission, he was given oxygen inhalation through a nasal cannula, and oral administration of 0.4 g of climycin, once a day. At the same time, the medication was the antihypertensive drug nifedipine sustained-release tablets 30mg, once a day, and valsartan capsules 80mg, once a day.
- the admission temperature is as high as 38°C, and the laboratory test results of the auxiliary examination result are as follows:
- Novel coronavirus nucleic acid test positive;
- Lung CT right pneumonia lesions, viral pneumonia is not excepted, and bilateral pleura is locally thickened.
- the patient took moxifloxacin orally for 3 days (400 mg QD) for anti-infection, nebulized interferon for 16 days (5 million BID), oral ribavirin for 10 days (150 mg TID), and 10 days of antiviral and antiviral abidol.
- Chinese herbal medicine treatment During the hospitalization period, Divudine and Efavirenz were administered regularly to treat HIV. Clinical symptoms improved: the patient had no cough and sputum, no chest tightness, shortness of breath, and normal body temperature. Lung CT showed no significant progress, and continued treatment for 26 days. The patient's sputum new coronavirus nucleic acid test continued to be positive. Orally administered 0.4g of climycin, once a day, and the sputum and throat swabs on the 2nd and 3rd days of the medication were all negative for the new coronavirus. The patient had no discomfort during the treatment.
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Abstract
本发明公开了酰基化螺旋霉素在治疗冠状病毒疾病药物中的应用;本发明采用计算机模拟证明可利霉素主要活性成分之一异戊酰螺旋霉素I与冠状病毒S蛋白及其宿主细胞受体ACE2蛋白结合;显示异戊酰螺旋霉素I具有潜在的ACE2抑制活性;冠状病毒感染后会导致ACE2表达减少,进而导致肺损伤增多;异戊酰螺旋霉素I与ACE2结合后,一方面可以抑制ACE2的功能,另一方面可以增加ACE2的稳定性,能抑制冠状病毒感染后ACE2表达减少的情况;异戊酰螺旋霉素I还可以与冠状病毒主要蛋白酶Mpro抑制位点发生强结合,抑制冠状病毒的转录和复制;体外和体内实验都表明可利霉素及其主要活性成分能够抑制冠状病毒及导致的疾病。
Description
本发明涉及抗生素、特别是大环内酯类抗生素或者异戊酰螺旋霉素类化合物或者其组合的新用途,具体地,本发明涉及酰基化异戊酰螺旋霉素类似物或者其组合在制备治疗冠状病毒感染疾病药物上的应用。
异戊酰螺旋霉素类似物为中国医学科学院医药生物技术研究所生物工程室王以光教授研究组应用同源重组技术将碳霉素4”-异戊酰转移酶基因整合至螺旋霉素产生菌(Streptomyces spiramyceticus F21)染色体上构建的基因工程菌所产生的一系列抗生素;作为科技部重大专项,该杂合抗生素为国家一类新药,通用名可利霉素;是中国医学科学院医药生物技术研究所与沈阳同联集团有限公司共同开发的国家1类创新药。可利霉素为多组分小分子结构:包括异戊酰螺旋霉素Ⅲ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅰ三个主要成分及一定量的丁酰、丙酰、乙酰螺旋霉素Ⅲ、丁酰、丙酰、乙酰螺旋霉素Ⅱ等9种成分组成的混合化学物。
异戊酰螺旋霉素Ⅰ:分子式:C48H82N2O15分子量:926.00
异戊酰螺旋霉素Ⅱ:分子式:C50H84N2O16分子量:968.00
异戊酰螺旋霉素Ⅲ:分子式:C51H86N2O16分子量:982.00
其主要成分结构如下:
上式中的Ⅰ、Ⅱ、Ⅲ即为异戊酰螺旋霉素化合物Ⅰ、Ⅱ、Ⅲ。
螺旋霉素的作用机制是特异性地与细菌核糖体50S亚基结合,通过阻止新生肽链的延伸而干扰细菌蛋白质的合成。螺旋霉素在体内的降解产物是脱碳霉糖后的降解产物活性降低或消失,而4"位酰化后,变为脱福洛氨糖降解途径,形成仍有抗菌活性的普拉特霉素A1等产物。4”异戊酰化侧链还能提高其体内的稳定性,延迟水解,延长半衰期的药代动力学的特点。研究表明螺旋霉素4”位酰化时,包括对某些大环内酯类抗生素耐药菌在内,其体内抗菌活性增强。
研究表明异戊酰螺旋霉素类似物抗菌活性强,对支原体和衣原体也有显著的抑制活性,与同类药物无明显交叉耐药性。除对耐药的革兰阳性菌(如耐甲氧西林金黄色葡萄球菌和耐药的化脓性链球菌)等有效外,对一些产β-内酰胺酶的细菌也有很好的疗效,对部分革兰阴性菌(如艰难梭菌、流感杆菌)以及真菌类的白色念珠菌也有较好的活性。
由于该异戊酰螺旋霉素类似物具有毒性低、剂量小、服药次数少和服用方便等优点,是在适应证范围内患者追求安全、有效药物的首选;开发其潜在的新用途也具有显著的意义。
冠状病毒属的病毒是具外套膜(envelope)的正链单股RNA病毒,直径约80~120nm,其遗传物质是所有RNA病毒中最大的,只感染人、鼠、猪、猫、犬、禽类脊椎动物。
1975年,国家病毒命名委员会正式命名了冠状病毒科。到目前为止,大约有15种不同冠状病毒株被发现,能够感染多种哺乳动物和鸟类,有些可使人发病。
2019年末至2020年1月,部分地区发生了不明原因肺炎,患者支气管肺泡灌洗标本送检新一代测序,发现新型冠状病毒,全基因组序列与造成SARS流行的蝙蝠来源冠状病毒序列一致性超过85%;该新型冠状病毒(SARS-CoV-2)毒株分离培养,转染人气道上皮细胞后观察到细胞病变,胞外可见游离病毒颗粒和胞质膜囊内充满病毒颗粒的包涵体,进一步证实该冠状病毒为第7种可感染人类的冠状病毒家族成员。
截止2020年1月29日,全国新型冠状病毒肺炎,确诊人数超过6000人、疑似人数9000多人,造成死亡100多人。
1月25日凌晨,NEJM在线发表武汉不明原因肺炎病原学确证的原创性研究及观点文章,指出2019-nCoV是过去20年来在人类中出现的第三种冠状病毒。其他两种能够导致传染性非典型肺炎(SARS)和中东呼吸综合征(MERS)的冠状病毒曾大规模爆发,在感染者中造成很高病死率,而且目前尚无特异性抗冠状病毒药物或疫苗被证实对人类有效。这些基因组序列研究将促进抗病毒药物、疫苗以及实验动物模型的研发。
2020年2月12日,WHO将该新型冠状病毒(SARS-CoV-2)导致的疾病命名为:Corona Virus Disease 2019,COVID-19,目前2019-nCoV肺炎确诊病例仍在增加,疫情防控形式依然严峻,寻找有效的治疗药物、特别是已经上市的药物用于治疗COVID-19刻不容缓。
发明内容
本发明的第一个目的在于提供一种酰基化螺旋霉素类化合物或者其组合物在制备治疗冠状病毒感染疾病药物中的应用;特别是异戊酰螺旋霉素类化合物或者组合物在冠状病毒感染疾病药物中的应用;具体地,可利霉素在治疗冠状病毒感染疾病药物中的应用。
本发明的第二个目的在于提供酰基化螺旋霉素类化合物及其组合物在制备治疗2019-nCoV冠状病毒感染疾病药物中的应用;特别是异戊酰螺旋霉素类化合物及其组合物在治疗2019-nCoV冠状病毒感染疾病药物中的应用。具体地,可利霉素在治疗2019-nCoV病毒导致的疾病药物中的应用。
本发明的第三个目的在于提供酰基化螺旋霉素类化合物或者其组合物在制备治疗冠状病毒感染导致的并发症药物中的应用;特别是异戊酰螺旋霉素类化合物或者组合物在治疗冠状病毒感染疾病导致的并发症药物中的应用;具体地,可利霉素在治疗冠状病毒疾病导致的并发症药物中的应用。具体地,可利霉素在治疗2019-nCoV肺炎导致的并发症药物中的应用。
[根据细则91更正 11.12.2020]
为了实现上述目的,本发明采用的技术方案为:
为了实现上述目的,本发明采用的技术方案为:
酰基化螺旋霉素类化合物或者其组合物在制备治疗冠状病毒感染疾病药物中的应用;特别是异戊酰螺旋霉素类化合物或者其组合物在冠状病毒感染疾病药物中的应用;具体地,可利霉素在治疗冠状病毒感染疾病药物中的应用。
酰基化螺旋霉素类化合物及其组合物在制备治疗冠状病毒感染疾病药物中的应用;特别是异戊酰螺旋霉素类化合物及其组合物在治疗2019-nCoV疾病药物中的应用。具体地,可利霉素在治疗2019-nCoV疾病药物中的应用。
抗生素、特别是大环内酯类抗生素化合物在制备治疗冠状病毒感染疾病等疾病药物中的应用,特别是2019-nCoV感染疾病药物中的应用。
本发明中所述的酰基化酰螺旋霉素类化合物至少为选自由基因工程方法制备可利霉素得到的异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ三个主要成分之一、及一定量的丁酰、丙酰、乙酰螺旋霉素Ⅲ、丁酰、丙酰、乙酰螺旋霉素Ⅱ等9种成分之一或者其组合。优选为选自异戊酰螺旋霉素类化合物,更优选异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ之一、其结构类似物或者其中至少两种的组合、或者可利霉素,其中可利霉素为国家一类新药,当然包括任何化学或者生物方法得到的上述化合物或者其组合物,或者可利霉素的类似物。
本发明所述的冠状病毒包括α属和β属冠状病毒,优选:HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63、HCoV-HKU1、MERS-CoV,和2019-nCoV,优选SARS-CoV、MERS-CoV,和2019-nCoV,其中SARS为严重急性呼吸综合征、MERS为中东呼吸综合征、以及2019-nCoV(COVID-19)新型冠状病毒感染。
本发明所述的冠状病毒感染疾病包括呼吸道、消化道和神经系统疾病,包括但不限于如感冒、发烧、额窦炎、中耳炎、咽炎、慢性支气管炎、肺炎、胸腔积液、各种呼吸综合征、急性肠胃炎、心肺疾病、免疫力低下、重复感染、肺损伤、器官衰竭等疾病。特别是SARS、MERS以及2019-nCoV导致的各种疾病及并发症,包括各种感染性疾病。
本发明的酰基化螺旋霉素类化合物或者其组合物与冠状病毒主要蛋白酶Mpro抑制位点结合。
本发明的酰基化螺旋霉素类化合物或者其组合物与冠状病毒S蛋白及其宿主细胞受体ACE2蛋白结合。
特别是本发明的异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ或者可利霉素与冠状病毒S蛋白及其宿主细胞受体ACE2蛋白结合。
特别是本发明的异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ或者可利霉素与冠状病毒主要蛋白酶Mpro抑制位点结合。
本发明所述的治疗药物包括本发明的酰基化螺旋霉素类化合物之一、或者至少两种的其组合、或者可利霉素作为第一药物活性成分,辅助以第二药物活性成分,所述的第二药物活性成分可以选自抗病毒药物、和/或抗艾滋病药物,第一药物活性成分和第二药物活性成分可以为独立制剂,也可以复配为一种制剂。
优选本发明的异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ之一或者其至少两种的组合、或者可利霉素作为第一药物活性成分,与第二药物活性成分进行组合。
所述的第二药物活性成分可以是蛋白酶抑制剂、融合蛋白抑制剂、核苷类逆转录酶抑制剂、免疫抑制剂等,选自以下药物活性成分的至少一种:茚地那韦(Indinavir)、沙奎那韦(Saquinavir)、洛匹那韦(Lopinavir)、卡非佐米(Carfilzomib)、瑞托那韦(ritonavir)、瑞德西韦或伦地西韦(Remdesivir RDV,GS-5734)、阿扎那韦、达芦那韦、替拉那韦、福沙那韦、思扎托韦、普瑞托韦、利巴韦林、阿巴卡韦、硼替佐米、埃替格韦、孟鲁司特、脱氧土大黄苷、虎杖苷、山豆根查耳酮、双硫仑、卡莫氟、紫草素、依布硒、Tideglusib、PX-12、TDZD8、肉桂硫胺、环孢菌素A、艾博卫泰、氨溴索、干扰素、阿比多尔、奥司他韦等药物或者药物中的活性成分。
下面结合附图对本发明的具体实施方式作进一步详细的描述。
附图作为本发明的一部分,用来提供对本发明的进一步的理解,本发明的示意性实施例及其说明用于解释本发明,但不构成对本发明的不当限定。
图1是本发明异戊酰螺旋霉素I与血管紧张素转换酶ACE2结合示意图之一;
图2是本发明异戊酰螺旋霉素I与血管紧张素转换酶ACE2结合示意图之二;
图3是本发明异戊酰螺旋霉素I与spike蛋白结合示意图;
图4是SARS-CoV主要蛋白酶(Mpro,又称3CL蛋白酶)的3D结构及其抑制剂结合位点示意图,即在介导病毒复制和转录功能方面的作用示意图;
图5是本发明异戊酰螺旋霉素I和3CL的结合示意图;
图6是几种不同药物与SARS-CoV Mpro的对接(docking);
图7可利霉素抑制CCR5表达示意图;
图8为异戊酰螺旋霉素I(CM926)与SARS-S系统柔性分析示意;
图9为异戊酰螺旋霉素I(CM926)与SARS-S结合示意图;(A)SARS-S与CM926的2D结合模式;(B)CM926在SARS-S分子表面的结合模型;(C)SARS-S与CM926的3D结合模式;
图10为异戊酰螺旋霉素I(CM926)与SARS-M系统柔性分析;
图11为异戊酰螺旋霉素I(CM926)与SARS-M结合示意;(A)SARS-M与CM926的2D结合模式;(B)CM926在SARS-M分子表面上的结合模型;(C)SARS-M与CM926的3D结合模式;
图12为异戊酰螺旋霉素I(CM926)与ACE2系统柔性分析;
图13为异戊酰螺旋霉素I(CM926)与ACE2结合示意(A)ACE2与CM926的2D结合模式;(B)CM926在ACE2分子表面上的结合模型;(C)ACE2与CM926的3D结合模式;
图14为异戊酰螺旋霉素I(CM926)与ACE2-SARS系统柔性分析;
图15为异戊酰螺旋霉素I(CM926)与复合示意ACE2-SARS结合示意,(A)ACE2-SARS与CM926的2D结合模式;(B)CM926在ACE2-SARS分子表面上的结合模型;(C)ACE2-SARS与CM926的3D结合模式;
图16可利霉素在Huh 7.5细胞中对HCoV-229E毒株诱导的CPE的抑制作用;
图17可利霉素抗炎及免疫调节-抑制PI3K/Akt/mTOR通路;
图18可利霉素可以明显降低肺部损伤;
图19患者可利霉素治疗前后肺部炎症改善之一;
图20患者可利霉素治疗前后肺部炎症改善之二;
图21是具体案例中病例1的患者可利霉素治疗前后肺部炎症改善情况。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对实施例中的技术方案进行清楚、完整地描述,以下实施例用于说明本发明,但不用来限制本发明的范围。
计算机模拟实验:
参见附图2,异戊酰螺旋霉素I为可利霉素主要活性物质之一,也是可利霉素的特征性成分之一;经分子对接,显示异戊酰螺旋霉素I可以与spike-ACE2蛋白复合物在多个位点发生结合。异戊酰螺旋霉素I与ACE2具有强结合,且结合位置位于ACE2活性位点口袋内(图1)。该结构与ACE2催化功能有关,ACE2发挥催化功能时需要改变该区域的三维结构,而异戊酰螺旋霉素I的结合会限制ACE2的三维结构变化,因此异戊酰螺旋霉素I具有ACE2抑制作用。异戊酰螺旋霉素化合物Ⅱ、Ⅲ与spike蛋白也结合,结合的位置位于ACE2活性位点口袋内(图略)。
冠状病毒的S蛋白(spike)组合成一个三聚体,约含有1300个氨基酸,属于第一类膜融合蛋白(Class I viral fusion protein),同类的病毒膜融合蛋白还包括HIV的Env蛋白,流感的HA蛋白,以及埃博拉病毒的Gp蛋白等。S蛋白决定了病毒的宿主范围和特异性,也是宿主中和抗体的重要作用位点。图3a、3b显示异戊酰螺旋霉素I在spike蛋白RBD结构域附近也有结合,因此异戊酰螺旋霉素I可能会抑制SARS病毒与宿主受体的结合。预示着本发明的戊酰螺旋霉素I能够抑制SARS病毒或流感病毒。异戊酰螺旋霉素化合物Ⅱ、Ⅲ与spike蛋白也结合,因此异戊酰螺旋霉素化合物Ⅱ、Ⅲ或者可利霉素能够抑制SARS病毒或流感病毒。
2020年1月21日,中国科学家(科学院上海巴斯德研究所郝沛研究员、军事医学研究院国家应急防控药物工程技术研究中心钟武研究员和中科院分子植物卓越中心合成生物学重点实验室李轩研究员)在SCIENCE CHINA Life Sciences(《中国科学:生命科学》英文版),在线发表了题为“Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission”的论文,通过对新型冠状病毒SARS-CoV-2基因组与2002年“非典”SARS冠状病毒、“中东呼吸综合征”MERS冠状病毒进行了全基因组比对,发现平均分别有~70%和~40%的序列相似性,其中不同冠状病毒与宿主细胞作用的关键spike基因(编码S-蛋白),有更大的差异性。发现新型冠状病毒SARS-CoV-2属于Beta冠状病毒属(Betacoronavirus)。Betacoronavirus是蛋白包裹的单链正链RNA病毒,寄生和感染高等动物(包括人)。在进化树的位置上,与SARS(导致2002年“非典”)病毒和类SARS(SARS-like)病毒的类群相邻,但并不属于SARS和类SARS病毒类群;进化上共同的外类群是一个寄生于果蝠的HKU9-1冠状病毒。文章作者利用分子结构模拟的计算方法,对冠状病毒SARS-CoV-2的S-蛋白和人ACE2蛋白进行了结构对接研究,获得了令人惊讶的结果。虽然冠状病毒SARS-CoV-2的S-蛋白中与ACE2蛋白结合的5个关键氨基酸有4个发生了变化,但变化后的氨基酸,却整体性上非常完美的维持了SARS病毒S-蛋白与ACE2蛋白互作的原结构构象。尽管新型冠状病毒SARS-CoV-2的新结构与ACE2蛋白互作能力,由于丢失的少数氢键有所下降(相比SARS病毒S-蛋白与ACE2的作用有下降),但仍然达到很强的结合自由能(-50.6kcal/mol)。这一结果说明新型冠状病毒SARS-CoV-2是通过S-蛋白与人ACE2互作的分子机制,来感染人的呼吸道上皮细胞。SARS的S-蛋白与2019-nCoV病毒的S-蛋白结构,二者RBD 结构域的3D结构几乎相同,该结构域与宿主细胞受体ACE2直接相互作用。基于SARS的S-蛋白结构,对2019-nCoV病毒做了结构模拟,然后基于SARS与ACE2组成复合晶体的结构,评估2019-nCoV病毒S-蛋白与人类ACE2分子相互作用的能力。2019-nCoV病毒S-蛋白与人ACE2之间的结合自由能为-50.6kcal/mol,比SARS的-78.6kcal/mol高了28kcal/mol。相较而言,2019-nCoV病毒S-蛋白与人体ACE2的结合相对较弱,但是这个亲和力仍被认为是很强的。
分子模拟结果显示:可利霉素主要活性成分之一异戊酰螺旋霉素I与SARS-CoV S蛋白及其宿主细胞受体ACE2蛋白都有结合,且对后者的结合作用极强。
异戊酰螺旋霉素I和ACE2蛋白的结合会限制ACE2的三维结构变化,显示异戊酰螺旋霉素I具有潜在的ACE2抑制活性。SARS感染后会导致ACE2表达减少,进而导致肺损伤增多;基于小分子与蛋白结合后可增强蛋白稳定性,异戊酰螺旋霉素I与ACE2结合后,一方面可以抑制ACE2的功能,另一方面可以增加ACE2的稳定性,有可能抑制SARS感染后ACE2表达减少的情况。ACE2在人角膜、结膜均有表达,目前已有通过角膜、结膜感染2019-nCoV病毒的报道,进一步证明2019-nCoV病毒是通过S-蛋白与人ACE2相互作用,来感染人的呼吸道上皮细胞。异戊酰螺旋霉素I对冠状病毒S蛋白及其宿主细胞受体ACE2均可能具有抑制作用,因此可能对冠状病毒感染具有抑制作用,与其结构类似的异戊酰螺旋霉素Ⅲ、异戊酰螺旋霉素Ⅱ、及丁酰、丙酰、乙酰螺旋霉素Ⅲ、丁酰、丙酰、乙酰螺旋霉素Ⅱ等可利霉素的活性成分以及可利霉素具有潜在的对冠状病毒感染具有抑制作用,特别是对2019-nCoV病毒感染具有抑制作用。
图4表明SARS-CoV主要蛋白酶(Mpro),又称3CL蛋白酶,在介导病毒复制和转录功能方面发挥着关键作用。结构域I和结构域II之间为抑制剂的结合部位。
图5显示异戊酰螺旋霉素I和3CL的结合位点与3CL抑制剂的结合位点一致,Mpro(3CL)已被中科院上海药物所用于分子模拟技术筛选冠状病毒药物的筛选模型,并于2020.1.25日发布相关筛选结果。因此异戊酰螺旋霉素I在抗病毒感染方面具有极大的潜在价值。与其结构类似的异戊酰螺旋霉素Ⅲ、异戊酰螺旋霉素Ⅱ、及丁酰、丙酰、乙酰螺旋霉素Ⅲ、丁酰、丙酰、乙酰螺旋霉素Ⅱ等可利霉素的活性成分以及可利霉素在抗病毒感染方面具有极大的潜在价值,特别是对2019-nCoV病毒感染极大的潜在价值。
本发明还比较了几种不同药物与SARS-CoV Mpro的docking,参见图6和表1。可以看出,在这些药物中,异戊酰螺旋霉素I与SARS-CoV Mpro的docking明显强于其他药物,不仅优于同类药物,而且优于具有抗病毒作用的洛匹那韦和利托那韦。而其他临床常用抗生素不仅结合力弱于异戊酰螺旋霉素I,且不能与抑制位点完全结合,对Mpro很难起到有效抑制。异戊酰螺旋霉素I因其异戊酰基团的存在,可以完美地与Mpro抑制位点完全结合,预示异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅲ、异戊酰螺旋霉素Ⅱ、及丁酰、丙酰、乙酰螺旋霉素Ⅲ、丁酰、丙酰、乙酰螺旋霉素Ⅱ等可利霉素的活性成分以及可利霉素在冠状病毒感染、炎症反应以及继发性感染等方面均具有良好的临床价值。
表1几种不同药物与SARS-CoV M
pro的结合能
上述结果说明异戊酰螺旋霉素I、螺旋霉素I、阿奇霉素、红霉素、莫西沙星、克拉霉素等抗生素,以及大环内酯类抗生素具有一定的作用。可以成为治疗新型冠状病毒感染疾病的潜在药物。
目前新型病毒感染报道主要症状是免疫损伤反应导致间质性肺炎,多器官功能损伤、肺部损伤等,从前期实验观察到的可利霉素药效,虽说机制还有很多有待进一步明确,鉴于目前新型肺炎本身有合并细菌感染,且可利霉素药物安全性高,可利霉素可以治疗新型病毒感染。
进一步,本发明采用异戊酰螺旋霉素I与蛋白:SARS-Spike,SARS-Mpro,ACE2,ACE2-SARS-Spike分别进行对分对接,然后进行分子动力学模拟。
分子对接方法:
使用MOE软件进行分子对接。异戊酰螺旋霉素I(小分子CM926)在MOE中通过能量优化得到低能三维构象,作为配体。结合位点的定义如下:SARS-S(Cys323,Ser358),SARS-M(Glu166,Phe140,Cys145),ACE2(Lys74,Ser106,Gln102,Asp350),ACE2-SARS(Asp38,Gln42,Gln325,Glu329,Arg426,Tyr436)。在正式对接之前,选择AMBER10:EHT力场以及R-field隐式溶剂模型。对接流程采用柔性的induced fit模式,结合口袋氨基酸的侧链可根据配体构象进行优化调整,约束侧链转动的权重设置为10。配体的结合模式首先通过London dG打分函数进行排序,前30个构象通过进一步优化和GBVI/WSA dG方法对结合自由能再次评价。选择打分最佳(结合自由能最低)的结合模式,进行分子动力学模拟。
分子动力学模拟
选择MMFF94x力场生成小分子异戊酰螺旋霉素I的参数。异戊酰螺旋霉素I的氢原子在HF/6-31g*下由Gaussian09软件包优化。并用HF/6-31g*进行电荷计算,再用RESP4拟合的方法拟合静电势。在复合物中加入钠/氯抗衡离子使体系中和,并在TIP3P的长方形水盒子中溶剂化,使水盒子边缘和溶质表面之间形成
的溶剂层。
所有的分子动力学模拟均在AMBER16软件中进行。复合物中加入钠/氯抗衡离子,使体系维持电中性。应用AMBER GAFF和FF14SB力场,并使用SHAKE算法限制所有涉及共价键的氢原子,时间步长为2fs。采用粒子网格Ewald(PME)方法处理长程静电相互作用。在加热步骤之前对每个溶剂化系统进行两次能量优化步骤。最初的4000步能量优化是在所有重原子被
的能量限制下进行的,而溶剂分子和氢原子可以自由移动。然后进行非约束能量优化,包括2000步最陡下降优化和2000步共轭梯度优化。再使用Langevin动力学,在恒定体积条件下,将整个系统在50ps时间内从0K加热至300K,然后在1atm的恒定压力下模拟400ps。在加热步骤中,使用
的弱能量约束来限制所有重原子。在动力学模拟的平衡阶段,采用周期性边界,并在NPT(恒定成分,压力和温度)条件下(1atm和300K的恒定压力)进行20ns模拟。最后使用MM-PBSA方法计算复合物的结合自由能。
如图8所示,蛋白SARS-S骨架原子的RMSD值小于2.5埃,同时异戊酰螺旋霉素I(小分子CM926)的RMSD值小于5.0埃,整个体系在模拟时间内达到了平衡,说明体系模拟时采用的力场是合适的。对动力学模拟得到的轨迹进行聚类分析,并取体系稳定后的轨迹的聚类中心作为最终的异戊酰螺旋霉素I与蛋白SARS-S的结合模式构象,如图9所示。
异戊酰螺旋霉素I位于SARS-S的结合位点周围。然而,在异戊酰螺旋霉素I和SARS-S之间没有形成明显的氢键和Pi-Pi堆积相互作用。异戊酰螺旋霉素I的大部分暴露在溶剂中。CM926与结合位点周围残基之间形成了范德华相互作用。
如图10所示,蛋白SARS-M骨架原子的RMSD值小于2.5埃,同时小分子CM926的RMSD值小于3.0埃,整个体系在模拟时间内达到了平衡,说明体系模拟时采用的力场是合适的。对动力学模拟得到的轨迹进行聚类分析,并取体系稳定后的轨迹的聚类中心作为最终的小分子CM926与蛋白SARS-M的结合模式构象,如图11所示。
CM926与SARS-M的结合位点形成了适当的空间互补性。CM926和SARS-M之间形成了氢键和静电相互作用。被视为氢键供体的CM926羟基中的氧原子与Glu166的侧链氧原子形成氢键。CM926氨基中的氮原 子被视为氢键供体,分别与Glu166和Gln189的侧链氧原子形成氢键。氨基中带有正电荷的氮原子与Glu166带有负电荷的侧链氧原子形成静电相互作用。在CM926和周围残基之间形成了VDW相互作用。这些主要相互作用促进CM926与SARS-M蛋白之间的结合。
如图12所示,蛋白ACE2骨架原子的RMSD值小于3.0埃,同时小分子CM926的RMSD值小于3.0埃,整个体系在模拟时间内达到了平衡,说明体系模拟时采用的力场是合适的。对动力学模拟得到的轨迹进行聚类分析,并取体系稳定后的轨迹的聚类中心作为最终的小分子CM926与蛋白ACE2的结合模式构象,如图13所示。
CM926与ACE2的结合位点形成了适当的空间互补性。CM926和ACE2之间形成了氢键和静电相互作用。CM926羟基中的氧原子被视为氢键供体,与Asp206的侧链氧原子形成氢键。CM92氨基中的氮原子被视为氢键供体,分别与Asp206和Asp350的侧链氧原子形成氢键。CM926羰基中的氧原子被视为氢键受体,与Lys562的侧链氮原子形成氢键。氨基中带正电荷的氮原子分别与带有负电荷的Asp206和Asp350的侧链氧原子形成静电相互作用。在CM926和周围残基之间形成了VDW相互作用。这些主要相互作用促进CM926与蛋白质ACE2之间的结合。
如图14所示,蛋白ACE2-SARS骨架原子的RMSD值小于5.0埃,同时小分子CM926的RMSD值小于3.0埃,整个体系在模拟时间内达到了平衡,说明体系模拟时采用的力场是合适的。对动力学模拟得到的轨迹进行聚类分析,并取体系稳定后的轨迹的聚类中心作为最终的小分子CM926与蛋白ACE2-SARS的结合模式构象,如图15所示。
CM926与ACE2-SARS的结合位点形成了适当的空间互补性。CM926和ACE2-SARS之间形成了氢键和静电相互作用。CM926A氨基中的氮原子被视为氢键供体,与复杂蛋白中ACE2亚基的Glu35的侧链氧原子形成氢键。氨基中带正电荷的氮原子与Glu35带负电荷的侧链氧原子形成静电相互作用。在CM926和周围残基之间形成了VDW相互作用。这些主要相互作用促进CM926与蛋白质ACE2-SARS之间的结合。
CM926与蛋白的结合自由能ΔGtotal如表1所示。结合自由能由4部分组成,其中范德华作用能量项由ΔEvdw表示,静电作用能量项由ΔEele表示,溶剂的极性作用能量项由ΔGpolar表示,溶剂的非极性作用能量项由ΔGnonpolar表示。小分子CM926与蛋白SARS-S,SARS-M,ACE2及ACE2-SARS的结合自由能分别为:-3.26,-6.20,-9.16and-6.76kcal/mol。预测的近似IC50值由以下公式计算:
ΔGtotal≈RTlnIC50
其中,R为8.314,T为300K
最终预测的近似IC50值分别为1817.33μM,30.27μM,0.21μM及11.82μM。
表2 Average Binding Energy and its Components Obtained from the MM-PBSA Calculation for the complexes.
(由MM-PBSA计算获得的平均结合能及其组成)
通过分子动力学模拟实验,预测与异戊酰螺旋霉素I与SARS Mpro蛋白、ACE2蛋白及SARS Spike蛋白RBD结构域的抑制作用,其IC50值分别约为30.27μM,0.21μM和11.82μM,因此推测可利霉素在抑制新型冠状病毒(2019-nCoV)传染、抑制病毒复制等方面具有极大的应用价值。
体外实验
本发明对可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ分别进行了体外实验,验证其对冠状病毒的作用,采用细胞病变效应(CPE)实验测定在Huh7.5细胞、HCT-8细胞和MRC-5细胞中对冠状病毒(HCoV-229E和HCoV-OC43)的半数抑制浓度(IC
50)及SI。
材料
供试品:可利霉素,为沈阳同联集团有限公司提供。DMSO配成20mg/ml母液,4℃冰箱保存。实验时以培养液稀释至适当浓度后进行实验。4℃冰箱保存。
阳性对照药:利巴韦林注射液(RBV)购自天津金耀集团湖北天药药业股份有限公司,批号为31712252,规格为100mg/ml,用时稀释至所需浓度,4℃冰箱保存。
阿奇霉素:市售
细胞
传代人结肠癌细胞HCT-8细胞,人肝癌细胞Huh7.5细胞和人二倍体细胞MRC-5细胞均为本室(中科院生计所)传代保存,在含10%胎牛血清(inactivated fetal bovine serum)和1%双抗(青霉素和链霉素)的DMEM或1640培养基中,37℃,5%CO2培养箱中培养。2-3天传代一次。
HCoV-229E于Huh7.5细胞中传代,保存于-80℃冰箱。HCoV-OC43于HCT-8细胞中传代,保存于-80℃冰箱。
DMEM液体培养基、1640液体培养基、胎牛血清(fetal bovine serum)、青霉素和链霉素溶液(penicillin-streptomycin)、PBS(PH=7.4)和0.25%Trypsin-EDTA均购自Invitrogen公司。
实验用品及仪器:细胞培养瓶、96孔培养板和移液管为美国Corning公司产品;二氧化碳孵箱(Model3111)为美国Thermo公司产品;生物安全柜为美国NUAIRE公司产品;倒置显微镜,奥林巴斯公司产品;真空泵为INTEGRA Biosciences公司产品;12道移液器和单道移液器为Eppendorf产品。
实验方法:细胞培养以HCT-8细胞为例:在长满HCT-8细胞的培养瓶内加0.25%Trypsin-EDTA 3ml,37℃消化1-2分钟,弃消化液,加培养液吹打,1:4传代,2-3天传代一次,种板时配制成每毫升20万个细胞,接种96孔细胞培养板,每孔0.1ml,37℃,5%CO2培养过夜,细胞长成单层后进行实验。
抗HCoV-229E的活性测定(CPE法):实验在传代Huh7.5细胞中进行,Huh7.5细胞1×10
4个/孔接种于96孔板中,过夜培养后将100μl HCoV-229E病毒液感染96孔板内Huh7.5细胞,待测药物用维持液稀释,分别于感染同时给药和感染后2h给药两种给药方案进行测定,待测药物以三倍稀释8个剂量的样品进行实验,每个剂量设2个平行孔,待病毒对照组病变达4+号时观察结果,记录并用Reed-Muench法计算药物对病毒的半数抑制浓度(公式如下)及选择指数(SI=IC50/TC50)。
其中:A=累积抑制率<50%的药物浓度,B=累积抑制率>50%的抑制率,C=累积抑制率<50%的抑制率,D=log稀释倍数
实验结果
药物在Huh7.5细胞内对HCoV-229E的抑制作用
如表3所示,感染同时给药:CPE法测定可利霉素0h给药对HCoV-229E毒株的IC50为3.14±0.80μg/ml,选择指数SI为13.0;RBV在0h给药对HCoV-229E的IC50为7.06±0.91μg/ml,选择指数SI为>14.2;阿奇霉素在0h给药对HCoV-229E的IC50为16.78±3.48μg/ml,选择指数SI为5.0。
感染后2h给药:CPE法测定可利霉素2h给药对HCoV-229E毒株的IC50为2.36±0.30μg/ml,选择指数SI为15.1;RBV在2h给药对HCoV-229E的IC50为6.24±2.07μg/ml,选择指数SI为>16.0;阿奇霉素在2h给药对HCoV-229E的IC50为9.55±6.75μg/ml,选择指数SI为6.7。
表3:药物对Huh7.5细胞内HCoV-229E的抑制作用(IC
50)(CPE法)
药物对HCoV-OC43毒株的药效
如表4所示:在HCT-8细胞中,CPE法测定可利霉素0h给药对HCoV-OC43毒株的IC50为25.87μg/ml,选择指数SI为2.2;RBV在0h给药对HCoV-OC43的IC50为11.11μg/ml,选择指数SI为>9.0;阿奇霉素在0h给药对HCoV-OC43无抑制作用。
如表5所示:在MRC-5细胞中,CPE法测定可利霉素0h给药对HCoV-OC43毒株的IC50为5.93μg/ml,选择指数SI为3.2;RBV在0h给药对HCoV-OC43的IC50为63.75μg/ml,选择指数SI为>1.6;阿奇霉素在0h给药对HCoV-OC43无抑制作用。
表4:药物对HCT-8细胞内对HCoV-OC43的抑制作用(IC
50)(CPE法)
表5:药物对MRC-8细胞内对HCoV-OC43的抑制作用(IC
50)(CPE法)
图16可以看出可利霉素在Huh 7.5细胞中对HCoV-229E毒株诱导的CPE的抑制作用
总结:
在本次试验条件下:在Huh7.5细胞中,CPE法测定可利霉素0h给药对HCoV-229E毒株的IC50为3.14±0.80μg/ml,选择指数SI为13.0;RBV在0h给药对HCoV-229E的IC50为7.06±0.91μg/ml,选择指数SI为>14.2;阿奇霉素在0h给药对HCoV-229E的IC50为16.78±3.48μg/ml,选择指数SI为5.0。CPE法测定可利霉素2h给药对HCoV-229E毒株的IC50为2.36±0.30μg/ml,选择指数SI为15.1;RBV在2h给药对HCoV-229E的IC50为6.24±2.07μg/ml,选择指数SI为>16.0;阿奇霉素在2h给药对HCoV-229E的IC50为9.55±6.75μg/ml,选择指数SI为6.7。
在HCT-8细胞中,CPE法测定可利霉素0h给药对HCoV-OC43毒株的IC50为25.87μg/ml,选择指数SI为2.2;RBV在0h给药对HCoV-OC43的IC50为11.11μg/ml,选择指数SI为>9.0;阿奇霉素在0h给药对HCoV-OC43无抑制作用。
在MRC-5细胞中,CPE法测定可利霉素0h给药对HCoV-OC43毒株的IC50为5.93μg/ml,选择指数SI为3.2;RBV在0h给药对HCoV-OC43的IC50为63.75μg/ml,选择指数SI为>1.6;阿奇霉素在0h给药对HCoV-OC43无抑制作用。
上述实验可以得到如下结论:
本实验条件下,可利霉素对HCoV-OC43和HCoV-229E两个毒株均有抑制作用;RBV对HCoV-OC43和HCoV-229E两个毒株均有抑制作用;阿奇霉素对HCoV-229E毒株有抑制作用,对HCoV-OC43毒株没有抑制作用。在本实验条件下,可利霉素具有明确的体外抗冠状病毒HCoV-229E和HCoV-OC43活性,且优于阿奇霉素。RBV的抗冠状病毒HCoV-229E和HCoV-OC43活性与文献和本实验之前的结果相当,说明实验系统成立。
以上结果表明,可利霉素对2种病毒的药效均强于对照药物利巴韦林,同时强于大环内酯类抗生素阿奇霉素,说明其具有很好的抗冠状病毒活性,有很好的应用前景。
本发明采用上述方法检测了本发明的异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ,其中异戊酰螺旋霉素I的结果高于可利霉素,异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ接近可利霉素。
本发明还采用上述方法进行了可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ抑制中东呼吸综合症冠状病毒(MERS-CoV)、重症急性呼吸综合征冠状病毒SARS-CoV、人冠状病毒HKU1、人冠状病毒NL63(HCoV-NL63)的体外实验,结果均高于照药物利巴韦林或阿奇霉素。
特别是,本发明对可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ抗2019-nCoV活性进行了体外实验,具体如下:
受试药物:可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ。
细胞:VeroE6细胞,由中国医学科学院医学实验动物研究所病原中心保存。
病毒:2109-nCoV,滴度为105TCID50/ml,由中国医学科学院医学实验动物研究所病原中心-80℃保存。使用病毒滴度为100TCID50。
实验方法:
受试药物:可利霉素采用两个浓度:10μg/ml,2μg/ml分别进行实验。
表6.药物名称及使用浓度
受试药物抗病毒实验
(1)无菌96孔培养板,每孔加入200μl浓度为5×104cell/ml Vero E6细胞,37℃5%CO2培养24小时;
(2)受试药物稀释成2个浓度,每个浓度5个复孔,每孔100μl,然后每孔再加入等体积100TCID50病毒,作用1h;
(4)1h后,弃去96孔培养板中细胞培养液,加入上述混合液;
(5)同时设立细胞对照、空白对照(溶剂对照)和病毒对照(阴性对照);
(6)细胞37℃,5%CO2孵箱孵育4-5天;
(7)光学显微镜下观察细胞病变(CPE),细胞完全病变记录为“++++”,75%病变记录为“+++”,50%病变记录为“++”,25%病变记录为“+”,未病变记录为“-”。
实验条件:以上实验操作均在BSL-3实验室内完成。
结果判断:
细胞不出现CPE为有效抑制病毒的浓度,出现CPE为无效。
实验结果:
药物分别设置两个浓度,在细胞上均能有效抑制2019-nCoV复制,结果见表7.
表7.可利霉素抗2019-nCoV效果
结论:根据细胞水平的筛选结果,可利霉素在2μg/ml浓度下,可抑制2109-nCoV在细胞内复制,提示可利霉素具有体外抗2109-nCoV活性。
同样异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ也具有体外抗2109-nCoV活性。
可利霉素IC
50计算
表8
实际药物浓度 表9
其他实验:
可利霉素明显改善全身重要器官特别是肺、肝,肾和心,脑,胃,肠等器官炎症和水肿病理改变,对免疫缺陷裸鼠感染体内抗菌和改善炎症和水肿,可利霉素通过启动体内免疫机制起到抗菌和改善组织损伤的效果,对于非感染性炎症---可见炎症病灶减轻,疼痛减轻或消失,水肿消失等;可抑制PI3K/Akt/mTOR通路;在动物体内可以显著降低IL-1β、IL-4等免疫因子水平;有潜在的抑制M2型巨噬细胞,提高M1型巨噬细胞的功能;有极强的诱导M2型巨噬细胞向M1型巨噬细胞转化的功能,而M2型巨噬细胞促进炎症。
参见图17可利霉素和异戊酰螺旋霉素I、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ抗炎及免疫调节-抑制PI3K/Akt/mTOR通路,从而降低IL-6和IL-8水平示意。其中可利霉素处理A549细胞48h,可抑制mTOR及p-mTOR的表达,可利霉素可以抑制mTOR下游P70S6K1(核糖体蛋白S6激酶)以及p-P70S6K1的表达,研究表明mTOR受到抑制后,IL-6和IL-8水平会明显降低。
新型冠状病毒(2019-nCoV)与其他冠状病毒相同,会刺激患者的先天免疫系统,致使体内大量释放细胞因子,造成细胞因子风暴和急性炎症反应。这会导致全身血管更为脆弱,引发急性呼吸窘迫症和多器官衰竭。本发明的可利霉素具有抗病毒作用,可以在早期使用降低病毒对免疫系统的作用,对于重症患者,通过控制细胞因子风暴和急性炎症反应,参见图18,可利霉素可以明显降低肺及其他器官损伤。
图18为可利霉素对肺损伤的修复示意图,可利霉素在临床治疗剂量(200-2000mg/人/天)范围内,给药6天可以改善小鼠因感染导致的组织炎症反应和组织细胞损伤;HE病理染色显示对机体组织炎症和损伤改善更有效;说明可利霉素对有可能改善机体免疫系统功能,修复组织损伤,促进康复;
可利霉素同时具有安全性高,组织分布广,组织浓度高,半衰期长,退热作用明显的特点,同时具有抗炎、调节免疫、抗水肿等作用,对于耐药肺炎克雷伯菌及鲍曼不动杆菌均有较好的治疗效果,在抗击冠状病毒感染的战役中,具有潜在的临床应用价值。结合上述分子模拟计算结果,可以认为可利霉素在冠状病毒感染、炎症反应以及继发性感染等方面均具有良好的临床价值。
已经在多个医院通过伦理,进行临床试验证明抑制2019-nCoV的效果。
作为科技部重大专项,对口服可利霉素对新冠病毒肺炎(2019-nCoV)患者疗效和安全性的进行了随机、开放、阳性药对照、多中心临床研究。为临床有效、安全治疗提供重要科学依据。
中国境内北京、湖北、辽宁、黑龙江数个治疗2019-nCoV的指定医院参与临床研究。受试者年龄18-75周岁,符合新型冠状病毒感染肺炎诊断标准(第五版)。患者符合以下任何一条:(1)再次出现发热、或临床症状加重,(2)咽拭子核酸检测阴转阳性,(3)临床症状无改善或核酸持续阳性,(5)胸部CT显示肺炎症或纤维化进展。SOFA评分:1分-13分。
治疗方法
轻型:可利霉素片,每次0.4g,每日1次,餐后口服,连续用药7天,治疗结束后进入随访观察期30天。
普通型:可利霉素片,每次0.4g,每日1次,餐后口服,10天,治疗结束后进入随访观察期30天。
重型、危重型:可利霉素片,每次0.4g,每日1次,餐后口服,连续用药14天,不能口服者,鼻饲管给药。治疗结束后进入随访观察期30天。
主要疗效指标:1.热退时间(天)。2.肺部炎症消退时间(HRCT)(天)。3.治疗结束3天、7天咽拭子新冠病毒转阴率(%)。
次要疗效指标:1.临床症状及药物不良反应描述。2.用药后第1天、第3天、第5天、第7-10天外周血白细胞计数较基线的变化值。3.用药后第1天、第3天、第5天、第7-10天PCT、C反应蛋白较基线的变化值。4.用药后第7-10天、第14天胸部影像学特征较基线的变化情况。5.住院时间及病死率。
入组病例总体情况
病毒“复阳”或经治患者47例,包括轻型11例,普通型27例,重型3例,危重型6例。经前期治疗后,病毒核酸仍阳性的患者40例,核酸阴性的患者7例。
主要结果:
(一)主要疗效评价。
1.40例病毒核酸阳性的患者中,16例患者3天核酸阴转,13例患者7天阴转,1例患者15天阴转,后加入10例患者经治疗后均在15日之内阴转。
2.入组时有肺部炎症的患者19例(核酸阴性7例,核酸阳性12例),7天肺部炎症显著改善率为73.7%(14/19)。图19为其中一名患者可利霉素治疗前后肺部炎症改善,从图中可以看出可利霉素治疗5、10天肺部炎症明显改善;图20另一患者,(A)为患病第一天CT扫描图,(B)为患病第五天CT扫描图,(C)为患病第6天CT扫描图(开始可利霉素治疗日),(D)为患病第8天CT扫描;(E)为患病第11天CT扫描图。
3.入组时发热的患者5例(核酸阴性3例,核酸阳性2例),3天体温复常率为60%(3/5),7天体温复常率为100%(5/5)。
(二)次要疗效评价。
1.入组时有呼吸道症状患者11例(核酸阴性2例,核酸阳性9例),3天呼吸道症状消失率为36.4%(4/11),7天呼吸道症状消失率为100%(11/11)。
2.出院患者21例,治愈出院率为44.7%(21/47),1周内出院患者9例。
3.入组患者病情均未加重,无死亡病例。
(三)安全性评价。
尚未发现与药物相关的严重不良反应,仅有1例服用可利霉素第6天时出现一过性恶心,1例发现皮疹,停药后症状消失。
根据以上结果初步判断:针对病毒“复阳”或经治治疗失败患者,可利霉素可以快速、有效清除新冠病毒,显著改善临床症状与肺部炎症,可以提高新冠肺炎患者的预后,缩短住院时间。可利霉素临床应用方便,每天1次口服给药,无明显的不良反应。
具体案例分析
病例1:
患者,女性,72岁。因干咳、乏力,无发热,因接触史在新冠病毒肺炎定点医院隔离观察。后患者干咳加重,出现呼吸急促,曾服洛匹那韦/利托那韦片,每次400mg/100mg,每天2次,吸氧对症治疗,症状未缓解、出现呼吸困难入北京某指定医院。
实验室检查:咽拭子RT-PCR显示新冠病毒阳性,吸氧状态下氧饱和度83%,血气分析氧分压64mmHg,血常规检查正常,肝肾功能正常,既往高血压病史,一直服用降压药物,平时血压稳定。入院后给予鼻导管吸氧,口服可利霉素0.4g,每天1次。同时用药为降压药物硝苯地平缓释片30mg,每天1次,缬沙坦胶囊80mg,每天1次。入院后第2天患者一般状态好转,咳嗽、呼吸困难明显好转,氧饱和度提升到98%;血气分析氧分压升130mmHg,可利霉素治疗后第3天、第6天(2月25、27日)2次咽拭子核酸检测均阴性,CT(图21)显示病程第6天(服用可利霉素前1天),双侧肺纹理增多,右肺下野可见不规则毛玻璃样病灶,左侧散在斑片状影(A箭头所示);病程第9天(服用可利霉素后3天),双侧肺纹理清晰,右肺下野不规则毛玻璃样病灶明显吸收(B箭头所示),病程第12天(服用可利霉素后5天),右肺病灶明显吸收(C箭头所示)少量纤维化形成。无不良反应报告。住院治疗6天达到出院标准。出院后继续口服可利霉素至10天,目前随访无异常。
经验:1.本例老年患者疾病进展时发现及时,尽管伴随高血压等疾病,及时治疗后预后好;2.本例患者治疗很简单,除了可利霉素及常用降压药物,未应用其他药物,包括输液;3.患者依从性好。
病例2:
患者,女性,49岁,HIV 8年,一直服用抗艾滋病药物。高血压病史5年,未规律治疗。入院体温最高达38℃,辅助检查结果实验室检查结果:
1.血常规:常规检查2.甲流病毒抗原:阴性;
3.新型冠状病毒核酸检测:阳性;
4.肺CT:右肺炎性病变,病毒性肺炎不除外,双侧胸膜局限性增厚。
诊断:
1.新型冠状病毒感染肺炎(普通型);
2.艾滋病;
3.高血压病。
主要治疗情况:
患者入院后口服莫西沙星3天(400mg QD)抗感染、雾化干扰素16天(500万BID)、先后口服利巴韦林10天(150mg TID)、阿比多尔10天抗病毒及中草药治疗。住院期间一直规律口服双夫定、依非韦仑治疗HIV。临床症状好转:患者无咳嗽咳痰,无胸闷气短,体温正常。肺部CT无明显进展,持续治疗26天,患者痰新冠病毒核酸检测持续阳性。口服可利霉素0.4g,每天1次,用药第2天、3天痰及咽拭子新冠病毒检查均因阴性。治疗期间患者无不适。
病例3:
患者,男,41岁,因“胸闷、乏力5天”于2020年2月9日收入院。2月10日患者第二次咽拭子新冠病毒核酸检测阳性,诊断新型冠状病毒肺炎。2020年2月15日患者开始使用可利霉素片0.4g,qd,至今(2月23日)仍在使用。2020年2月16日(试用可利霉素片第1d)复查血常规提示淋巴细胞计数正常范围,心肌酶正常,未见明显心肌损伤。2020年2月17日(试用可利霉素片第2d)患者目前病情稳,活动后感胸闷,静息下呼吸尚平稳,鼻导管吸氧,胸部CT结果与前片(2020-2-9)比较,右肺上叶及中叶间质性病变范围较前减小,密度较前变淡。2月19日核酸结果阴性。2020年02月21日(试用可利霉素片第7d)患者病情稳定。2月22日(试用可利霉素片第8d)胸部CT右肺上叶及中叶间质性病变较前(2020-2-17)略有吸收。2月22日第二次核酸结果阴性。2月24日出院。治疗期间患者未发生明显的药物不良反应。
病例4:
患者,男,57岁,因“咳嗽1周”于2020年2月09日收入院治疗,2020.2.15日加重,同时检测新型冠状病毒核酸检测结果为阳性2020.2.19停用其他药物开始使用可利霉素片0.4g,qd。2020.2.20日(试用可利霉素片第2d)患者诉无咳嗽,无发热,心慌胸闷明显好转。2.25复查新型冠状病毒核酸检测阴性;复查胸部CT:双肺感染性病变较前部分吸收、好转。2020.2.27日复查复查新型冠状病毒核酸检测阴性。治疗期间未出现药物不良反应。
病例5:
患者,女,67岁,患者2020.2.3因发热入院,诊断为“新型冠状病毒肺炎(危重型)”,给予无创呼吸机辅助通气(2.6-2.19)、吸氧(2.19-2.25)、抗病毒药物治疗,2.24日复查床旁胸片提示双肺片状增高影,较前吸收不佳,淋巴细胞计数0.82×109/L,血气分析提示氧分压64mmHg,于2020.2.24日停用抗病毒药物,应用可利霉素治疗。2.26日复查胸部CT较前好转,2.27复查淋巴细胞计数1.09×109/L,血气分析提示氧分压升至正常。
病例6:
患者,女,69岁,患者2020.2.3因发热、胸闷、气喘入院,诊断为“新型冠状病毒肺炎(危重型)”治疗,给予综合治疗,患者体温正常核酸复查阴性,2.24日复查床旁胸片提示双肺感染,较前吸收不佳,临床表现:无发热,不断咳嗽,伴呼吸困难,持续经鼻高流量吸氧,氧浓度80%,氧流量45L/min于2020.2.24日开始服用可利霉素,入组后无发热,2.25持续经鼻高流量吸氧,氧浓度60%,氧流量35L/min,2.26氧浓度60%,氧流量30L/min,2.27氧浓度50%,氧流量30L/min,3.1复查床旁胸片较前好转,3.2复查胸部CT较前好转。
下表中列举了其他可利霉素治疗新冠临床研究的例子。
表10其他研究可利霉素治疗新冠临床研究
另外,可利霉素临床试用治疗新冠肺炎疗效明显。湖北某市医院及其所属区县医院32例患者(新增1例危重型患者试用)入院后应用《新冠病毒肺炎诊疗指南(第5版)》推荐的抗病毒药物基础上使用可利霉素400mg,qd。用药第3-5天,3例患者体温恢复正常,3例患者反复发热,26例使用可利霉素前无发热症状。经治抗病毒治疗失败的8例患者,用药第3-10天咽拭子2019nCOVRNA转阴率100%。
Claims (10)
- 抗生素、特别是大环内酯类抗生素化合物在制备治疗冠状病毒感染疾病等疾病药物中的应用。
- 根据权利要求1所述的应用,所述抗生素为酰基化螺旋霉素类化合物或者其组合物;优选异戊酰螺旋霉素类化合物或者其组合物在制备治疗冠状病毒感染疾病药物中的应用。
- 根据权利要求1所述的应用,所述冠状病毒感染疾病中的冠状病毒包括α属和β属冠状病毒;优选HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63、HCoV-HKU1、MERS-CoV,和/或2019-nCoV,更优选SARS-CoV、MERS-CoV,和/或2019-nCoV。
- 根据权利要求1至4任何一项所述的应用,冠状病毒感染疾病包括呼吸道、消化道和神经系统疾病,包括但不限于感冒、发烧、额窦炎、中耳炎、咽炎、慢性支气管炎、肺炎、胸腔积液、各种呼吸综合征、急性肠胃炎、心肺疾病、免疫力低下、重复感染、肺损伤、器官衰竭等疾病;特别是SARS、MERS以及2019-nCoV导致的各种疾病。
- 根据权利要求1至4任何一项所述的应用,其中酰基化螺旋霉素类化合物或者其组合物与冠状病毒主要蛋白酶Mpro抑制位点结合。
- 根据权利要求1至4任何一项所述的应用,其中酰基化螺旋霉素类化合物或者其组合物与冠状病毒S蛋白及其宿主细胞受体ACE2蛋白结合。
- 根据权利要求1至5任何一项所述的应用,其中冠状病毒感染疾病包括冠状病毒感染疾病导致的并发症。
- 权利要求1-8任何一项所述的应用,包括酰基化螺旋霉素类化合物之一、或者其组合、或者可利霉素作为第一药物活性成分,还包括第二药物活性成分,所述的第二药物活性成分可以选自抗病毒药物、和/或抗艾滋病药物,第一药物活性成分和第二药物活性成分可以为单独制剂,也可以是复配为一种制剂。
- 权利要求1-9任何一项所述的应用,所述的酰基化螺旋霉素类化合物或者组合物包括酰基化酰螺旋霉素类化合物之一、或者其组合、或者可利霉素作为第一药物活性成分,还包括第二药物活性成分,所述的第二药物活性成分为蛋白酶抑制剂、融合蛋白抑制剂、核苷类逆转录酶抑制剂、或者免疫抑制剂之一或者其组合。
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CN101511374A (zh) * | 2006-07-13 | 2009-08-19 | 埃科动物健康有限公司 | 泰伐洛星作为抗病毒剂的用途 |
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CN113940930A (zh) * | 2021-12-06 | 2022-01-18 | 西安交通大学 | 鼠李秦素在制备抗新型冠状病毒药物中的应用及药物 |
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CN111671762A (zh) | 2020-09-18 |
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EP4098265A4 (en) | 2024-03-13 |
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