CN113975278A

CN113975278A - Application of bromocriptine in preparation of product for treating African swine fever

Info

Publication number: CN113975278A
Application number: CN202111270260.7A
Authority: CN
Inventors: 赖仞; 靳林; 刘洋; 王菁菁
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-01-28
Anticipated expiration: 2041-10-29
Also published as: CN113975278B

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 African swine fever virus family and the genus African swine fever virus, and is the only arbovirus DNA virus at present. The African swine fever virus particle has a diameter of about 250nm, is in an icosahedral form, and has a multilayer structure including a nucleoid, a nucleocapsid, a double-layer inner membrane, a capsid, and an envelope obtained when the virus buds through a plasma membrane. The African swine fever virus pS273R protease is involved in the cleavage of the African swine fever virus polyproteins pp220 and pp62, a cysteine protease encoded by the African swine fever virus gene pS273R, belonging to the SUMO-1 specific protease family according to the division, having a molecular weight of 31 kDa. The pS273R protease is very critical to the maturation of the virion, and if the pS273R protease expression 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 eccentric position, thereby affecting the infection capacity of the virus, and therefore the pS273R protease is considered as a potential target point for designing and developing products for treating African swine fever virus.

At present, no effective product can effectively inhibit 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 the 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 electrophoresis result of the E.coli soluble lysate induced without IPTG, lane 3 being the electrophoresis result of the E.coli soluble lysate induced with 0.8mM IPTG, and the arrow indicating the electrophoresis band containing the pS273R protease; b: separating and purifying pS273R protease, and separating and purifying by Sephadex G-50 gel chromatography and cation exchange column, wherein a lane 1 is a Marker, a lane 2 is an SDS-PAGE electrophoresis result of the escherichia coli soluble lysate, and an arrow indicates an electrophoresis band containing the pS273R protease;

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 Ki for inhibition of pS273R protease by Bromoccriptine.

Detailed Description

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 μ M.

The invention also provides application of bromocriptine in preparing a product for treating African swine fever.

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 regarded as an important antiviral target. Bromocriptine can inhibit the activity of African swine fever virus and treat African swine fever by obviously inhibiting the pS273R protease of the African swine fever virus.

In the invention, the molecular formula of bromocriptine is C₃₂H₄₀BrN₅O₅(ii) a The chemical structural formula of the bromocriptine is shown as a formula I;

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 on the market. In the practice of the present invention, bromocriptine is available from Master of Small Molecules.

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 (figure 1) searched in the NCBI Gene database, and then a recombinant plasmid pS273R/pET-28b (+) is constructed and transformed into an Escherichia coli strain BL-21(DE3) 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 important for packaging African Swine Fever Virus (ASFV) and has been regarded as an important antiviral target. Bromoccriptine (Bromocriptine, molecular formula: C)₃₂H₄₀BrN₅O₅) 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 broncriptine can inhibit pS273R protease. A Lineweaver-Burk plot of the inhibition of enzyme activity of 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, and the enzyme inhibition constants, i.e., Ki values, for each tick defensin were calculated by the Dixon method, and the inhibition constant Ki for the enzyme by Bromoccriptine 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 research institute of Chinese academy of sciences

<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

1. Use of bromocriptine for the preparation of a product inhibiting the activity of an enzyme of the SUMO-1 protease family.

2. The use of claim 1, wherein the enzyme in the SUMO-1 protease family comprises pS273R protease.

3. The use of claim 2, wherein the amino acid sequence of the pS273R protease is shown in SEQ ID No. 1.

4. Application of bromocriptine in preparing product for treating African swine fever is provided.

5. Application of bromocriptine in preparing products for inhibiting activity of African swine fever virus.

6. The use according to any one of claims 1 to 5, wherein the product comprises a medicament, vaccine, reagent or kit.