CN113975278B - Application of bromocriptine in preparation of product for treating African swine fever - Google Patents

Application of bromocriptine in preparation of product for treating African swine fever Download PDF

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CN113975278B
CN113975278B CN202111270260.7A CN202111270260A CN113975278B CN 113975278 B CN113975278 B CN 113975278B CN 202111270260 A CN202111270260 A CN 202111270260A CN 113975278 B CN113975278 B CN 113975278B
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protease
swine fever
bromocriptine
african swine
ps273r
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CN113975278A (en
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赖仞
靳林
刘洋
王菁菁
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Kunming Institute of Zoology of CAS
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides application of bromocriptine in preparation of a product for treating African swine fever, and belongs to the technical field of biological medicines. Bromocriptine can obviously inhibit pS273R protease of African swine fever virus, and can be used for preparing products for treating African swine fever.

Description

Application of bromocriptine in preparation of product for treating African swine fever
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of bromocriptine in preparation of a product for treating African swine fever.
Background
African Swine Fever (ASF) is an acute, hemorrhagic, virulent infectious disease caused by African Swine Fever Virus (ASFV) infection of domestic and wild pigs. The African swine fever virus belongs to the family of African swine fever virus and the genus African swine fever virus, and is the only arbovirus DNA virus at present. The African swine fever virus has a particle diameter of about 250nm, an icosahedral shape, and a multilayer structure including a nucleoid, a nucleocapsid, a double-layered inner membrane, a capsid, and an envelope obtained when the virus buds through a plasma membrane. The pS273R protease of African swine fever virus, a cysteine protease encoded by the African swine fever virus gene pS273R, is involved in the cleavage of the polyprotein pp220 and pp62 of African swine fever virus, and belongs to the SUMO-1 specific protease family according to the division, and has a molecular weight of 31kDa. The pS273R protease is very critical to the maturation of the virus particles, and if the pS273R protease is inhibited, the processing of two polyprotein bodies, pp220 and pp62, is inhibited, so that the virus is assembled into irregular icosahedral particles, and the irregular icosahedral particles are represented by an abnormal nucleocapsid with uneven thickness and a nucleoid with an off-center position, so that the infection capacity of the virus is influenced, and therefore the pS273R protease is considered as a potential target point for designing and developing products for treating African swine fever virus.
Currently, no effective product is available to effectively inhibit the pS273R protease.
Disclosure of Invention
In view of the above, the invention aims to provide an application of bromocriptine in preparation of a product for treating African swine fever, and the bromocriptine can obviously inhibit pS273R protease of African swine fever virus and can be applied to preparation of a product for treating African swine fever.
The invention provides application of bromocriptine in preparing a product for inhibiting the activity of enzymes in a SUMO-1 protease family.
Preferably, the enzyme in the SUMO-1 protease family comprises pS273R protease.
Preferably, the amino acid sequence of the pS273R protease is shown as SEQ ID No. 1.
The invention provides application of bromocriptine in preparing a product for treating African swine fever.
The invention also provides application of bromocriptine in preparing a product for inhibiting the activity of African swine fever virus.
Preferably, the product comprises a pharmaceutical, vaccine, reagent or kit.
The invention provides an application of Bromocriptine (Bromoccriptine) in preparation of an inhibitor of pS273R protease, and the Bromocriptine can obviously inhibit the pS273R protease of African swine fever virus and can be applied to preparation of a product for treating African swine fever.
Drawings
FIG. 1 is a cDNA sequence encoding the pS273R protease;
FIG. 2 is a result of identifying the mass of a collected sample using SDS-PAGE gel electrophoresis, in which A: inducible expression of the pS273R protease, lane 1 being Marker, lane 2 being the result of electrophoresis of the E.coli soluble lysate not induced with IPTG, lane 3 being the result of electrophoresis of the E.coli soluble lysate after induction with 0.8mM IPTG, and the arrow indicating the electrophoretic band containing the pS273R protease; b: separating and purifying the pS273R protease, and separating and purifying the protease by Sephadex G-50 gel chromatography and a cation exchange column, wherein the electrophoresis result of an escherichia coli soluble lysate is SDS-PAGE, a Marker is shown in a lane 1, the electrophoresis result of the escherichia coli soluble lysate is shown in a lane 2, and an electrophoresis band containing the pS273R protease is shown in an arrow;
FIG. 3 is a graph of the kinetics of Bromoccriptine inhibition of African swine fever virus pS273R protease;
FIG. 4 shows Bromoccriptine as a competitive inhibitor of pS273R protease, and also shows the Ki of Bromoccriptine for inhibition of pS273R protease.
Detailed Description
The invention provides application of bromocriptine in preparing a product for inhibiting the activity of enzymes in a SUMO-1 protease family.
In the present invention, the enzymes in the SUMO-1 protease family preferably include pS273R protease, and the amino acid sequence of the pS273R protease is shown as SEQ ID No.1, specifically:
MSILEKITSSPSECAEHLTNKDSCLSKKIQKELTSFLEKKETLGCDSESCVITHPAVKAYAQQKGLDLSKELETRFKAPGPRNNTGLLTNFNIDETLQRWAIKYTKFFNCPFSMMDFERVHYKFNQVDMVKVYKGEELQYVEGKVVKRPCNTFGCVLNTDFSTGTGKHWVAIFVDMRGDCWSIEYFNSAGNSPPGPVIRWMERVKQQLLKIHHTVKTLAVTNIRHQRSQTECGPYSLFYRARLDNVSYAHFISARITDEDMYKFRTHLFRIA。
in the present invention, the bromocriptine is a competitive inhibitor; the enzyme inhibition constant Ki value of bromocriptine is 72.742. + -. 12.621. Mu.M.
The invention also provides application of bromocriptine in preparing a product for treating African swine fever.
The invention also provides application of bromocriptine in preparing a product for inhibiting the activity of African swine fever virus.
In the present invention, the product preferably includes a drug, a vaccine, an agent or a kit.
The pS273R protease is important for packaging African Swine Fever Virus (ASFV) and has been considered as an important antiviral target. Bromocriptine can inhibit the activity of African swine fever virus and treat African swine fever by obviously inhibiting pS273R protease of the African swine fever virus.
In the invention, the molecular formula of bromocriptine is C 32 H 40 BrN 5 O 5 (ii) a The chemical structural formula of the bromocriptine is shown as a formula I;
Figure BDA0003328492180000031
the source of bromocriptine in the invention is not particularly limited, and the bromocriptine can be prepared by adopting a conventional preparation method in the field or can be obtained by market. In the practice of the present invention, bromocriptine is available from Master of Small Molecules, inc.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1. Inducible expression of recombinant pS273R protease
The sequence number of the African swine fever virus pS273R protease is 22220340 (FIG. 1) queried in the NCBI Gene database, and then a recombinant plasmid pS273R/pET-28b (+) is constructed and transformed into E.coli strain BL-21 (DE 3) for prokaryotic expression of the pS273R protease. The recombinant protease was purified from the soluble fraction of E.coli lysates following induction of expression with 0.8mM IPTG. The recombinant protein was further purified by Ni-NTAHis Bind Resin nickel column and then His tag was cleaved by rTEV protease. After the product was separated and purified by Sephadex G-50 (hyperfine) dextran gel filtration column and cation exchange column (Cytiva), the mass of the collected sample was identified by SDS-PAGE gel electrophoresis, and the results are shown in FIGS. 2 and 3.
2. Inhibition test of Bromoccriptine on African swine fever virus pS273R protease
The pS273R protease is crucial to packaging of African Swine Fever Virus (ASFV), and has been considered as an important antiviral target. Bromoccriptine (Bromocriptine, molecular formula: C) 32 H 40 BrN 5 O 5 ) Concentration gradients of (4) were set at 0. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M and 100. Mu.M, 3 multiple wells for each concentration. Bromoccriptine and pS273R protease were incubated at 37 ℃ for 15min, substrate Bz-Nle-Gly-Gly-Arg-AMC was added at substrate concentrations of 100. Mu.M and 200. Mu.M, and the substrate was cleaved with pS273R and then fluoresced under excitation light at 360nm, and the resulting fluorescence was measured at 460nm using a microplate reader (BioTek instruments, inc., USA) and shown as Relative Fluorescence Units (RFU), and an enzyme reaction curve was generated using GraphPad Prism6 software. Results referring to fig. 3, it was shown that bromorphiptine can inhibit pS273R protease. A Lineweaver-Burk plot of the inhibition of enzyme activity of the pS273R protease by Bromoccriptine was made using Graphpad Prism6 software, and the results are shown in FIG. 4. FIG. 4 shows that Bromoccriptine is a competitive inhibitor of pS273R protease, calculated by the Dixon method for each tick defenseThe enzyme inhibition constant of biotin, i.e., the Ki value, and the inhibition constant Ki of Bromopcriptine for this enzyme was 72.742. + -. 12.621. Mu.M.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> Kunming animal institute of Chinese academy of sciences
Application of <120> bromocriptine in preparation of product for treating African swine fever
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 272
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ser Ile Leu Glu Lys Ile Thr Ser Ser Pro Ser Glu Cys Ala Glu
1 5 10 15
His Leu Thr Asn Lys Asp Ser Cys Leu Ser Lys Lys Ile Gln Lys Glu
20 25 30
Leu Thr Ser Phe Leu Glu Lys Lys Glu Thr Leu Gly Cys Asp Ser Glu
35 40 45
Ser Cys Val Ile Thr His Pro Ala Val Lys Ala Tyr Ala Gln Gln Lys
50 55 60
Gly Leu Asp Leu Ser Lys Glu Leu Glu Thr Arg Phe Lys Ala Pro Gly
65 70 75 80
Pro Arg Asn Asn Thr Gly Leu Leu Thr Asn Phe Asn Ile Asp Glu Thr
85 90 95
Leu Gln Arg Trp Ala Ile Lys Tyr Thr Lys Phe Phe Asn Cys Pro Phe
100 105 110
Ser Met Met Asp Phe Glu Arg Val His Tyr Lys Phe Asn Gln Val Asp
115 120 125
Met Val Lys Val Tyr Lys Gly Glu Glu Leu Gln Tyr Val Glu Gly Lys
130 135 140
Val Val Lys Arg Pro Cys Asn Thr Phe Gly Cys Val Leu Asn Thr Asp
145 150 155 160
Phe Ser Thr Gly Thr Gly Lys His Trp Val Ala Ile Phe Val Asp Met
165 170 175
Arg Gly Asp Cys Trp Ser Ile Glu Tyr Phe Asn Ser Ala Gly Asn Ser
180 185 190
Pro Pro Gly Pro Val Ile Arg Trp Met Glu Arg Val Lys Gln Gln Leu
195 200 205
Leu Lys Ile His His Thr Val Lys Thr Leu Ala Val Thr Asn Ile Arg
210 215 220
His Gln Arg Ser Gln Thr Glu Cys Gly Pro Tyr Ser Leu Phe Tyr Arg
225 230 235 240
Ala Arg Leu Asp Asn Val Ser Tyr Ala His Phe Ile Ser Ala Arg Ile
245 250 255
Thr Asp Glu Asp Met Tyr Lys Phe Arg Thr His Leu Phe Arg Ile Ala
260 265 270

Claims (4)

1. Use of bromocriptine in the manufacture of a product for inhibiting the activity of an enzyme of the SUMO-1 protease family;
the enzymes in the SUMO-1 protease family include pS273R protease;
the amino acid sequence of the pS273R protease is shown as SEQ ID No. 1.
2. The use according to claim 1, further comprising the use of bromocriptine in the manufacture of a product for the treatment of African swine fever.
3. The use of claim 1, wherein the use further comprises the use of bromocriptine in the manufacture of a product for inhibiting the activity of African swine fever virus.
4. The use according to any one of claims 1 to 3, wherein the product comprises a medicament, vaccine, reagent or kit.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107921113A (en) * 2015-08-25 2018-04-17 巴比塔·阿格拉沃尔 Immune regulation composite and its application method
CN113388040A (en) * 2020-03-13 2021-09-14 普莱柯生物工程股份有限公司 African swine fever virus chimeric protein, vaccine composition, preparation method and application thereof

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EP3581576B1 (en) * 2013-03-15 2022-01-26 Incyte Holdings Corporation Tricyclic heterocycles as bet protein inhibitors for use in the treatment of a proliferative disease in combination with a janus kinase inhibitor
TW201722966A (en) * 2015-10-29 2017-07-01 英塞特公司 Amorphous solid form of a BET protein inhibitor
WO2020021090A1 (en) * 2018-07-27 2020-01-30 Conzelmann Karl Klaus Conditionally cytotoxic agents
CA3149480A1 (en) * 2019-08-02 2021-02-11 Enterin, Inc. Human squalamine derivatives, related compositions comprising the same, and methods of using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107921113A (en) * 2015-08-25 2018-04-17 巴比塔·阿格拉沃尔 Immune regulation composite and its application method
CN113388040A (en) * 2020-03-13 2021-09-14 普莱柯生物工程股份有限公司 African swine fever virus chimeric protein, vaccine composition, preparation method and application thereof

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