Application of p-benzoquinone and/or p-benzoquinone derivative in preparation of novel coronavirus resistant medicine
阅读说明：本技术 对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用 (Application of p-benzoquinone and/or p-benzoquinone derivative in preparation of novel coronavirus resistant medicine ) 是由 颜世强 杨杰 何淑旺 王文笙 程中伟 胡醒 于 2021-08-19 设计创作，主要内容包括：本发明提供了对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用,属于生物医药技术领域。对苯醌和/或对苯醌衍生物能够抑制新型冠状病毒的3C样蛋白酶(3C-likeprotease,3CLP)和/或木瓜样半胱氨酸蛋白酶(papain-likecysteineprotease,PLP)活性,从而抑制新型冠状病毒的活性和增殖,缓解感染的宿主细胞内免疫失衡,进而达到治疗新型冠状病毒的效果。(The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines, and belongs to the technical field of biological medicines. The p-benzoquinone and/or the p-benzoquinone derivative can inhibit the activity of 3C-like protease (3 CLP) and/or papain-like cysteine protease (PLP) of the novel coronavirus, so that the activity and proliferation of the novel coronavirus are inhibited, the immune imbalance in infected host cells is relieved, and the effect of treating the novel coronavirus is achieved.)
1. Application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines.
2. Application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel medicaments for inhibiting the multiplication of coronaviruses.
3. Use of p-benzoquinone and/or p-benzoquinone derivatives for the preparation of novel enzyme inhibitors of coronaviruses.
4. Use of p-benzoquinone and/or p-benzoquinone derivatives for the preparation of novel inhibitors of 3C-like protease and/or papain-like cysteine protease of coronaviruses.
5. The use according to any one of claims 1 to 4, wherein the chemical structural formula of the p-benzoquinone derivative is shown as formula I:
wherein, R is1Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is2Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is3Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is4Is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl.
6. The use according to claim 5, wherein the p-benzoquinone derivative comprises 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone.
7. The use according to claim 6, wherein the p-benzoquinone derivative comprises one or more of 2-methyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone.
8. Use according to claim 7, wherein the p-benzoquinone derivative comprises 2-methyl-1, 4-p-benzoquinone and/or 2-phenyl-1, 4-p-benzoquinone.
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of a novel coronavirus resistant medicine.
Background
The quinone compounds are chemical components with quinoid structures and mainly include four types of benzoquinone, naphthoquinone, phenanthrenequinone and anthraquinone. Benzoquinones (benzoquinones) are classified into two categories, namely o-benzoquinone and p-benzoquinone, and because o-benzoquinone is structurally unstable, most of naturally occurring benzoquinone compounds are derivatives of p-benzoquinone.
Various biological activities of benzoquinone compounds are disclosed in the prior art, for example, ubiquinone (coenzyme Q class) widely exists as p-benzoquinone derivatives, which participate in vivo redox reactions, wherein coenzyme Q10 can be used for treating hypertension, heart disease and cancer. For another example, idebenone, also a p-benzoquinone derivative, which was developed and marketed by wutian drug industry corporation of japan in 1986, has an activating effect on mitochondrial function; improving brain function metabolism and brain dysfunction; can improve the utilization rate of glucose in brain and promote the generation of ATP; can improve metabolism of neurotransmitter 5-hydroxytryptamine in brain, and has strong effects of resisting oxidation and scavenging free radicals.
In the prior art, no report on the application of benzoquinone compounds in resisting novel coronavirus exists.
Disclosure of Invention
The invention aims to provide application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines, wherein the p-benzoquinone and/or p-benzoquinone derivatives can effectively inhibit the activities of 3C-like protease (3 CLP) and pawpaw-like cysteine protease (PLP) of novel coronaviruses and can be used for preparing novel coronavirus resistant medicines.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines.
The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of a novel medicine for inhibiting multiplication of coronavirus.
The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel enzyme inhibitors of coronavirus.
The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel 3C-like protease and/or pawpaw-like cysteine protease inhibitors of coronavirus.
Preferably, the chemical structural formula of the derivative of p-benzoquinone is shown in formula I:
wherein, R is1Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is2Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is3Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl;
the R is4Is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl.
Preferably, the p-benzoquinone derivative includes 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone.
Preferably, the p-benzoquinone derivative comprises one or more of 2-methyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone.
Preferably, the p-benzoquinone derivative comprises 2-methyl-1, 4-p-benzoquinone and/or 2-phenyl-1, 4-p-benzoquinone.
The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines. The p-benzoquinone and/or the p-benzoquinone derivative can inhibit the activity of 3CLP and PLP of the novel coronavirus, so that the activity and proliferation of the novel coronavirus are inhibited, the immune imbalance in infected host cells is relieved, and the effect of treating the novel coronavirus is achieved.
Detailed Description
The invention provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines.
The invention also provides application of the p-benzoquinone and/or the p-benzoquinone derivative in preparing a novel medicine for inhibiting the multiplication of the coronavirus.
The invention also provides application of the p-benzoquinone and/or the p-benzoquinone derivative in preparation of a novel coronavirus enzyme inhibitor.
The invention also provides application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel inhibitors of 3C-like protease and/or papain-like cysteine of coronavirus.
In the present invention, the chemical structural formula of the derivative of p-benzoquinone is preferably as shown in formula I:
wherein, R is1Is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenylA substituted phenyl group; the R is2Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl; the R is3Selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl; the R is4Is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or poly-substituted phenyl.
In the present invention, the p-benzoquinone derivative preferably includes 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, one or more of 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ); more preferably, the p-benzoquinone derivative comprises one or more of 2-methyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone; most preferably, the p-benzoquinone derivative comprises 2-methyl-1, 4-p-benzoquinone and/or 2-phenyl-1, 4-p-benzoquinone.
The present invention is directed to p-benzoquinone (1, 4-p-benzoquinone), 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, the sources of 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone are not particularly limited and are derived from conventional commercial sources.
Polyproteins (PP) PP1a and PP1b are important for maintaining coronavirus RNA synthesis, and polyprotein maturation requires a series of cleavage processes to generate a multi-subunit protein complex (known as "viral replication transcriptase"). The major proteases (maprotease, Mpro) of the multi-subunit protein complex are 3C-like protease (3C-like protease, 3CLP) and papayaki-like cysteine protease (PLP), 3CLP and PLP are highly coordinated to catalyze the cleavage of polyprotein, and loss of 3CLP and PLP activity leads to the cessation of coronavirus life cycle.
In the present invention, the p-benzoquinone and/or p-benzoquinone derivative can inhibit the activity of 3CLP and PLP of the novel coronavirus, thereby inhibiting the activity and proliferation of the novel coronavirus, and relieving immune imbalance in infected host cells. In the present invention, the benzoquinone derivative exhibiting an inhibitory activity against 3CLP and PLP depends on the 1, 4-p-benzoquinone nucleus.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Expression and purification of novel coronavirus 3CLPro protein
1.1 primer design
Gene-specific primers (3 CL-F: gtgccgcgcggcagccatatgtcggcagtgctgcaaagcggtttccgtaagatg, SEQ ID NO: 1; 3 CL-R: gtggtggtggtggtgctcgaggggcccttgaaaggtcacaccgctgc, SEQ ID NO: 2) were designed for templates based on the gene of the novel coronavirus 3CLPro (protein residue 3264-3569 of ORF1ab, GenBank accession No.: MN908947.3), wherein the 3CLPro-F primer sequence carries an NdeI cleavage site and the 3CLPro-R primer sequence carries an XhoI cleavage site. Adding 5 amino acids of SAVLQ at the N end of the protein to construct a 3CLPro self enzyme cutting site; and adding two amino acids of GP behind the VTFQ amino acid at the C end of the protein to construct a Precission protease enzyme cutting site and promote the fusion expression of the target gene and the His label.
1.2 novel coronavirus 3CLPro gene amplification
A novel coronavirus 3CLPro gene having codon preference for Escherichia coli was first synthesized by a general-purpose organism. The total amplification system was 100. mu.L using the gene of the synthesized novel coronavirus 3CLPro as a template. The PCR reaction system comprises: cDNA template 1. mu.L, 3 CL-R4. mu.L, 3 CL-F4. mu.L, 2 XPPhanta MasterMix 50. mu.L and ddH2O41. mu.L. The reaction procedure is specifically as follows: pre-denaturation at 94 ℃ for 3min, 30 amplification reactions (94 ℃ for 15s, 65 ℃ for 30s, 72 ℃ for 30s) and full extension at 72 ℃ for 5 min. And taking 9 mu L of PCR product, adding 1 mu L of 6 xgel Loading Dye Purple, mixing uniformly, and then Loading, wherein 2 mu L of DNAmarker is added into the first glue hole for comparing the separated strip of the sample (voltage is 150V, and 40 min). The PCR results were observed under an ultraviolet lamp and bands of the correct fragments of the PCR were cut from the gel with a rubber cutter, placed in a 1.5mL EP tube, weighed and recorded. The PCR product was recovered using a SanPrep column DNA gel recovery kit, and the concentration and purity of the recovered target gene were determined using a Nano Drop.
1.3 construction of novel coronavirus 3CLPro expression vector
pET28a (+) was digested with NdeI and XhoI restriction enzymes, and the desired plasmid fragment was gel recovered for recombinant ligation. The specific connection system comprises: 20. mu.g of pET28a (+) plasmid fragment, 40. mu.g of 3CLPro target gene, 5. mu.M of 5 XCEII Buffer, 5. mu. L, Exnase II 2. mu.L, and make up to 50. mu.L with water.
1.4 inducible expression of novel coronavirus 3CLPro
The E.coli strain successfully transformed was inoculated in 25mL of LB liquid medium containing kanamycin (50. mu.g/mL) at 37 ℃ in an inoculum size of 1% (v/v) for preculture overnight, and then the culture was inoculated in 1L of LB liquid medium containing kanamycin (50. mu.g/mL) in an inoculum size of 1% (v/v) for culture at 37 ℃. When OD600 reached 0.8, 3CLPro protein was expressed by addition of 0.5mM isopropyl-D-thiogalactoside (IPTG) for 12h induction at 25 ℃.
1.5 purification of novel coronavirus 3CLPro
1) 1L of the bacterial liquid is centrifuged for 10min under the condition of 5000 Xg, and the supernatant is discarded.
2) The pellet was resuspended in 40mL Lysis Buffer (pH of all buffers was adjusted at 4 ℃) and then lysed by high pressure homogenizer for 3-5 min.
3) The lysate was ultracentrifuged (15000 Xg) at 4 ℃ for 30min using a cryo-centrifuge, and the supernatant was again ultracentrifuged (15000 Xg) for 30 min.
4) Prior to protein purification using a Ni-NTA column, the Ni-NTA column was equilibrated with a lysine Buffer.
5) Adding the supernatant of the lysate which is centrifuged for 2-3 times into a Ni-NTA column in batches for flowing through, after the flowing through is finished, adding about 100mL lysine Buffer to wash the Ni-NTA column in order to remove non-specific binding protein, and eluting by using 25mL of LWash Buffer and 35mL of Elution Buffer to collect 3 CLPro.
6) 20. mu.L of each of the lysate, flow-through, wash, Wash Buffer and Elution Buffer was collected.
7) mu.L of 5 SDS-PAGE Protein Loading Buffer was added to each sample and mixed well, the Protein samples were denatured by heating at 95 ℃ for 10min, 10. mu.L of each tube was subjected to SDS-PAGE electrophoresis, and 2. mu.L of precipitated Protein Ladder was added to the first gel well for control of the bands separated from the samples (voltage 220V, 45 min).
8) The eluate was concentrated to 2mL according to the SDS-PAGE electrophoresis result, and the eluate was replaced 3 times with replacement solution A (20mM HEPES, 400mM NaCl, pH 7.5) to finally obtain 4mL of 3CLPro concentrate, and the protein concentration was determined using Nano Drop. To the concentrate was added 13mg/mL of Precission Protease (PPE) in an amount of 100. mu.L overnight.
9) A GFC Buffer (100mM NaCl, 20mM HEPES, pH 7.5) equilibrated gel filtration molecular sieve chromatography column (HiLoad 16/600Superdex 200pg) was used.
10) 4mL of the 3CLPro concentrate after the digestion treatment were dispensed into 3 1.5mL EP tubes, and then ultracentrifuged at 12000 Xg at 4 ℃ for 10 min.
11) After removing the precipitate, the protein sample was pushed into a 5mL loading loop using a screw syringe and protein purification was performed by AKTA protein purifier.
12) The fraction containing high purity 3CLPro eluted from the gel filtration molecular sieve column was concentrated, and the concentrate was substituted 3 times with substitution solution B (20mM HEPES, 200mM NaCl, pH 7.5) and substitution solution C (20mM HEPES, 100mM NaCl, pH 7.5) in this order, and the protein concentration was determined using Nano Drop. Subpackaging 3CLPro protein at 50 μ L/tube, quick freezing with liquid nitrogen, and storing at-80 deg.C.
2. Expression purification of novel coronavirus PLpro
2.1 primer design
Gene-specific primers (PLP-F: GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAA, SEQ ID NO: 3; PLP-R: GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAAGCGGTTCGTACCATCAAG, SEQ ID NO: 4) were designed for the template based on the gene sequence of the novel coronavirus PLPro, wherein the PLP-F primer sequence carries NdeI cleavage sites and the PLP-R primer sequence carries EcoRI cleavage sites. Similarly, adding 5 amino acids of SAVLQ at the N end of the protein to construct a 3CLPro self enzyme cutting site; a stop codon was added at the C-terminus of the protein to allow for late termination of translation.
2.2 novel coronavirus PLPro Gene amplification
A novel coronavirus PLPro gene having codon preference for E.coli was first synthesized by a general-purpose organism. The total amplification system was 100. mu.L using the gene of the synthesized novel coronavirus PLPro as a template. The PCR reaction system comprises: cDNA template 1. mu.L, 3 CL-R4. mu.L, 3 CL-F4. mu.L, 2 XPphanta Master Mix 50. mu.L and ddH2O41. mu.L. The reaction procedure is specifically as follows: pre-denaturation at 94 ℃ for 3min, 30 amplification reactions (94 ℃ for 15s, 65 ℃ for 30s, 72 ℃ for 30s) and full extension at 72 ℃ for 5 min. And taking 9 mu L of PCR product, adding 1 mu L of 6 xgel Loading Dye Purple, mixing uniformly, and then Loading, wherein 2 mu L of DNA Marker is added into the first glue hole for controlling the separated strip of the sample (voltage is 150V, and 40 min). The PCR results were observed under an ultraviolet lamp and bands of the correct fragments of the PCR were cut from the gel with a rubber cutter, placed in a 1.5mL EP tube, weighed and recorded. The PCR product was recovered using a SanPrep column DNA gel recovery kit, and the concentration and purity of the recovered target gene were determined using a Nano Drop.
2.3 construction of novel coronavirus PLPro expression vector
pET28a (+) was digested with NdeI and EcoR I restriction enzymes, and the desired plasmid fragment was gel recovered for recombinant ligation. The specific connection system comprises: 20. mu.g of pET28a (+) plasmid fragment, 40. mu.g of PLPro target gene, 5 XCE II Buffer 5. mu. L, Exnase II 2. mu.L, and make up to 50. mu.L with water.
2.4 inducible expression of novel coronavirus PLPro
Transferring the successfully constructed escherichia coli into a conical flask filled with 100mL of LB liquid culture medium, adding 50mg/mL ampicillin antibiotic corresponding to the thallus according to the volume of 0.1% of the culture medium, placing the mixture into a shaking table at 37 ℃ for constant-temperature culture for about 2-3 h, adding 1 mu M ZnCl2 when the OD600 value of the bacterial liquid reaches 0.8, adding an inducer IPTG according to the final concentration of 0.5mM, and inducing overnight at 25 ℃ to express the protein.
2.5 purification of novel coronavirus PLPro
(1) Centrifuging the bacterial liquid under 4000 Xg for 15min, and discarding the supernatant. The pellet was resuspended at a ratio of 1L of the Lysis Buffer to 20mL (containing 5 mM. beta. -mercaptoethanol as a final concentration in the Lysis Buffer). And then cracking for 10min under the condition of 70-80 MPa by using a high-pressure homogenizer. The lysate was ultracentrifuged (18000 Xg) at 4 ℃ for 30min using a cryo-centrifuge and the procedure was repeated.
(2) After balancing the Ni-NTA column by using a lysine Buffer, adding the lysate supernatant obtained by twice centrifugation into the Ni-NTA column after membrane filtration, allowing the lysate supernatant to flow through the Ni-NTA column, adding the lysine Buffer to wash the Ni-NTA column to remove non-specifically bound protein, and eluting by using an Elution Buffer after washing to collect PLpro. 20. mu.L of lysate, flow-through solution, high-salt eluent, washing solution and Elution Buffer in the above experiment were collected, and 5. mu.L of 5 XSDS-PAGE protein loading Buffer was added to each. And (3) performing SDS-PAGE electrophoresis on 10 mu L of sample under 220V for 45min, dyeing and decoloring the protein gel after the electrophoresis is finished, and detecting whether the protein is expressed or not.
(3) The protein was concentrated according to the concentration of the protein eluted from the nickel column. GFC Buffer (20mM HEPES, 100mM NaCl, pH 7.5) was used to replace the eluate 3 times, and finally PLpro concentrate was obtained, and the protein concentration was determined using Nano Drop. The concentrate was digested overnight with 1M TCEP 5. mu.L and 51mg/mL 3CLpro protease 3. mu.L.
(4) A GFC Buffer equilibrium gel filtration molecular sieve chromatography column (HiLoad 16/600Superdex 200pg) was used. The digested PLpro concentrate was ultracentrifuged (12000 Xg) for 10 min. Protein samples were added to the loading loop using a screw syringe, taking care not to aspirate the pellet. The protein was purified twice using AKTA. The fraction containing high purity PLpro eluted from the gel filtration molecular sieve chromatography column was concentrated. Protein concentration was determined using Nano Drop. And (3) subpackaging the PLpro according to the requirements of subsequent experiments, quickly freezing by using liquid nitrogen after subpackaging, and storing at-80 ℃.
3. Activity assay
A compound: 1, 4-p-benzoquinone, 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 6-di-tert-butyl-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, idebenone and tetramethyl-1, 4-p-benzoquinone; the compounds described above are available from Chengdu Ji' an Technical Co., Ltd.
In the present invention, the p-benzoquinone (1, 4-p-benzoquinone), 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 6-di-tert-butyl-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-benzoquinone, 2, 6-dimethyl-1, 4-p-benzoquinone, 2, 6-benzoquinone, 2, 5-methyl-benzoquinone, 2, 4-benzoquinone, p-benzoquinone, and 2,5, 2, or 2, or 2, one, or a mixture of each, one, or a mixture, and a mixture, one, each, one, and one, each, one, each, one, each, one, each, one, The chemical structural formulas of 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, idebenone and tetramethyl-1, 4-p-benzoquinone are shown in table 1.
TABLE 1 structural formulas of p-benzoquinone and derivatives of p-benzoquinone
1) Test of inhibitory Activity of Compounds on novel coronavirus 3CLPro
(1) The activity of the novel coronavirus 3CLPro and the inhibitory activity of the compound on the novel coronavirus 3CLPro are measured by Fluorescence Resonance Energy Transfer (FRET) technology. Fluorescent substrates (Dabcyl-KTSAVLQ ↓ SGFRKM-E (Edans) -NH) with novel coronavirus 3CLPro cleavage sites (arrows) were used in the assay2) And Tris-HCl buffer (50mM Tris-HCl, 1mM EDTA, pH 7.3).
(2) Compounds were dissolved by 100% DMSO. mu.L of the above-mentioned compound was incubated with 40. mu.L of the novel coronavirus 3CLPro (final concentration: 0.1. mu.M) in Tris-HCl buffer for 1h, and 50. mu.L of fluorogenic substrate (Dabcyl-KTSAVLQ ↓: SGFRKM-E) (Edans) -NH2Final concentration of 20. mu.M) to initiate the reaction. A Dabcyl fluorescence signal (340nm (excitation)/490 nm (emission)) generated by cleavage of a substrate catalyzed by 3CLPro was detected using a radio resonance energy transfer fluorescence spectrophotometer, and the change in the fluorescence signal within 3min was used to determine whether or not the compound had an inhibitory activity.
(3) The kinetic constants for the novel coronavirus 3CLPro were derived by fitting the data to the MichaelisMenten equation. Screening out high inhibitory activity compounds (selecting compounds with inhibition rate of more than 0.9 at final concentration of 50 μ M) by primary screening, adding alkannin (known high activity inhibitor) as positive control, and comparing the inhibition rates. The screening results are shown in Table 2. From table 2, it can be seen that the compounds: 1, 4-p-benzoquinone, 2-methyl-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, 2-tert-butyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-methyl-1, 4-benzoquinone, 2, 5-dichloro-1, 4-benzoquinone, 2, 6-dichloro-1, 4-benzoquinone, 2, 6-p-benzoquinone, 2,6, 2, p-benzoquinone, 2, p-benzoquinone, or, The enzyme inhibition activity of tetrachloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone on the novel coronavirus 3CLPro is superior to that of the positive control medicament alkannin.
(4) The IC50 was determined for the high inhibitory activity compounds screened through the primary screen. Compounds were diluted to gradient concentrations (1.5625-100. mu.M and 0.15625-10. mu.M) using Tris-HCl buffer and assayed using the novel coronavirus 3CLPro and fluorogenic substrate system at the same final concentrations described above. IC50 values for compounds were calculated by GraphPad Prism 8.0 software. Triplicate experiments were performed and the final results are expressed as mean. + -. Standard Deviation (SD). The results are shown in Table 3. As can be seen from table 3, the compounds: 1, 4-p-benzoquinone, 2-methyl-1, 4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1, 4-p-benzoquinone, 2-bromo-1, 4-p-benzoquinone, thymoquinone, the IC50 values of 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethyl-1, 4-p-benzoquinone, 2-chloro-5-methyl-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrabromo-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone for the novel coronavirus 3CLPro were less than 1.0 μ M, and the compounds showed excellent inhibitory activity against the novel coronavirus 3 CLPro.
2) Test of inhibitory Activity of Compounds on novel coronavirus PLpro
(1) The activity of the novel coronavirus PLpro and the inhibitory activity of the compound on the novel coronavirus PLpro were determined using Fluorescence Resonance Energy Transfer (FRET) technique. The assay uses a fluorogenic substrate (CBE-RLRGG ↓AMC) with protein specific cleavage sites (indicated by arrows) to initiate the reaction. The protein and fluorogenic substrate were diluted with PLpro buffer (150mM NaCl, 40mM Tris-HCl, 5mM DTT, pH 8.0).
(2) Compounds were dissolved by 100% DMSO. mu.L of the compound was incubated with 40. mu.L of the novel coronavirus PLpro (final concentration: 0.3. mu.M) in PLpro buffer for 10min, and the reaction was initiated by adding 50. mu.L of fluorogenic substrate (final concentration: 20. mu.M). PLpro cleaves the substrate to generate AMC fluorescence signal (340nm (excitation)/490 nm (emission)), and the change in fluorescence signal within 3min is used to determine whether the compound has inhibitory activity.
(3) The PLpro kinetic constants for the novel coronavirus were obtained by fitting the data to the MichaelisMenten equation. Screening out high inhibitory activity compounds (selecting compounds with inhibition rate of more than 0.9 at final concentration of 50 μ M) by primary screening, adding alkannin (known high activity inhibitor) as positive control, and comparing the inhibition rates. The screening results are shown in Table 2. From table 2, it can be seen that the compounds: the enzyme inhibition activity of 2-methyl-1, 4-p-benzoquinone, thymoquinone, 2, 5-dimethyl-1, 4-p-benzoquinone, 2, 6-dimethoxy-1, 4-p-benzoquinone, 2, 5-diphenyl-1, 4-p-benzoquinone, 2, 5-dichloro-1, 4-p-benzoquinone, 2, 6-dichloro-1, 4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1, 4-p-benzoquinone and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone on the novel coronavirus PLPro is superior to that of the positive control shikonin. However, 2, 6-di-tert-butyl-1, 4-p-benzoquinone, idebenone and tetramethyl-1, 4-p-benzoquinone have an accelerating effect on the enzymatic activity of the novel coronavirus PLPro.
(4) The IC50 was determined for the high inhibitory activity compounds screened through the primary screen. Compounds were diluted to gradient concentrations using PLpro buffer and assayed using the same final concentration of SARS-CoV-2 PLpro and fluorogenic substrate system described above.
IC50 values for compounds were calculated by GraphPad Prism 8.0 software. Triplicate experiments were performed and the final results are expressed as mean. + -. Standard Deviation (SD). The results are shown in Table 3. As can be seen from Table 3, the compounds 2-methyl-1, 4-p-benzoquinone and 2-phenyl-1, 4-p-benzoquinone exhibited excellent inhibitory activity against the novel coronavirus PLPro with an IC50 value of less than 1.0. mu.M.
TABLE 2 Primary screening inhibition of p-benzoquinone or derivatives of p-benzoquinone against neocoronal activity of 50 μm
TABLE 3 measurement results of anti-neocrown Activity IC50 of p-benzoquinone or derivatives of p-benzoquinone
Note: "-" indicates that the compound of Table 2 was screened for relatively low activity and no IC50 determination was made; "+" indicates that the difference is large and cannot be obtained.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shandongdin Marine biopharmaceutical Co., Ltd
BEIJING DAYIN HIGH-TECH CHILDREN MEDICINE RESEARCH INSTITUTE Co.,Ltd.
<120> application of p-benzoquinone and/or p-benzoquinone derivatives in preparation of novel coronavirus resistant medicines
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