CN107823629A - Using application of the tannic acid as a series of compounds of representative in the infection of the Alphaviruses such as anti-chikungunya - Google Patents

Using application of the tannic acid as a series of compounds of representative in the infection of the Alphaviruses such as anti-chikungunya Download PDF

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Publication number
CN107823629A
CN107823629A CN201710908773.3A CN201710908773A CN107823629A CN 107823629 A CN107823629 A CN 107823629A CN 201710908773 A CN201710908773 A CN 201710908773A CN 107823629 A CN107823629 A CN 107823629A
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acid
infection
chloromethoxazole
oritavancin
application
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杨海涛
王赛男
刘祥
王泽方
陈成
蔡岩
郑金姣
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • 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
    • A61K31/65—Tetracyclines
    • 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
    • A61K31/7024—Esters of saccharides
    • 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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
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  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention provides using application of the tannic acid as a series of compounds of representative in the infection of the Alphaviruses such as anti-chikungunya, including tannic acid, chloromethoxazole sulfosalicylic acid, 2, the application of 3 dimercaptosuccinic acids and oritavancin diphosphonic acid in the infection of the Alphaviruses such as anti-chikungunya, available for chikungunya virus nsP2 protease (protease of NS2 Protein) activity is suppressed, it is expected to turn into the potential drug of the Alphaviruses such as anti-CHIKV infection.

Description

A series of compounds using tannic acid as representative infect in Alphaviruses such as anti-chikungunyas In application
Technical field
The present invention relates to the technical field of pharmacy, in particular it relates to tannic acid, chloromethoxazole sulfosalicylic acid, 2, The application of 3- dimercaptosuccinic acids and oritavancin diphosphonic acid in the infection of the Alphaviruses such as anti-chikungunya.
Background technology
Chikungunya fever is communicable disease caused by chikungunya virus infection.Chikungunya virus (chikungunya virus) abbreviation CHIKV mainly bites propagation by yellow-fever mosquito can cause heating, arthralgia, headache, skin Rash, myalgia wherein arthralgia and heating are that CHIKV infects most common symptom, and arthralgia being capable of periods of months Even the longer time brings serious health threat to patient.
Chikungunya fever isolates the virus in nineteen fifty-three in nineteen fifty-two Tanzania's the first explosion.“chikungunya” This name comes from local local, colloquial expressions, and it is curved for describing that arthralgia caused by CHIKV forces patient to have to mean " bends " Bent body.From 2 months 2005, a wide range of popular generation of Chikungunya fever was in each archipelago in the Indian Ocean.United according to the World Health Organization Meter, from 2005, India, Indonesia, Maldives, Burma and Thailand were it has been reported that more than 1,900,000 cases.By In April, 2015, Caribbean island, Latin American countries and the United States of America have recorded more than 1379788 Chikungunya fevers Suspected case.The disease also creates 191 death in the same period.Chikungunya fever eruption and prevalence lasting for a long time, to the mankind Grave danger be present in health.But do not treat the medicine or vaccine of Chikungunya fever at present.
CHIKV (Chikungunya Virus) belongs to Togaviridae alphavirus, is spherical, the single-stranded positive of coating RNA virus.Its Genome Size is about 12kb, including two ORFs parts, by 4 non-structural proteins (nsP1, NsP2, nsP3, nsP4) and 5 structural proteins part (C, E3, E2,6K, E1) compositions.Wherein nsP2 contains multiple functional areas, bag Include 1：N terminal domain；2：RecA-like domain 1A；3：RecA-like domain 2A；4：Protease； 5：The duplication of MTL domain, wherein nsP2 protease (protease of NS2 Protein) to virus plays important work With.Virus is in a replication process firstly the need of by the non-structural protein nsP1, nsP2, nsP3 of poly, nsP4 cracking, so as to complete The duplication of virus.Wherein, the non-structural protein cracking of poly is all that virus is cracked and then completed under nsP2 protease effects Replicate.Therefore CHIKV nsP2 protease also turn into a crucial antiviral drugs target, so for CHIKV's NsP2 protease carry out the inhibitor screening medicament research and development related to CHIKV and had a very big significance.
But so far, tannic acid, chloromethoxazole sulfosalicylic acid, DMSA and oritavancin two Phosphoric acid, the application in the infection of the Alphaviruses such as anti-CHIKV have no report.
The content of the invention
The problem of in correlation technique, the invention provides tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- bis- The application of dimercaptosuccinic acid and oritavancin diphosphonic acid in the infection of anti-chikungunya virus.
Present invention also offers tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and Ao Liwan Application of the star diphosphonic acid in the infection of anti-Alphavirus.
In above-mentioned application, tannic acid, chloromethoxazole sulfosalicylic acid, DMSA and oritavancin Diphosphonic acid is the micromolecular inhibitor of chikungunya virus nsP2 protease (protease of NS2 Protein).
In above-mentioned application, the structural formula of tannic acid is：
In above-mentioned application, the structural formula of chloromethoxazole sulfosalicylic acid is：
In above-mentioned application, the structural formula of DMSA is：
In above-mentioned application, the structural formula of oritavancin diphosphonic acid is：
Tannic acid CAS involved in the present invention is 1401-55-4, chloromethoxazole sulfosalicylic acid CAS is 73816-42-9, DMSA CAS are 304-55-2, oritavancin diphosphonic acid CAS is 192564-14-0, All compounds are bought in Shanghai Tao Su biochemical technologies Co., Ltd.By setting up negative control, tannic acid, chloromethane alkene are found Terramycin sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid are to the Alphavirus nsP2 such as CHIKV protease There is good inhibitory activity, therefore these compounds are expected to turn into the potential drug of the Alphaviruses such as anti-CHIKV infection.
Present invention also offers treat or prevent Alphavirus infection potential drug, its active component be respectively tannic acid, Chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid.The potential drug includes above-mentioned point Not or combination contains tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid One or more and pharmaceutically acceptable carrier.
In above-mentioned potential drug, the carrier includes diluent, excipient, filler, adhesive, wetting agent, disintegration Agent, sorbefacient, surfactant, absorption carrier, the one or more of lubricant and synergist.
And the preparation of the potential drug includes injection, tablet, pill, capsule, suspending agent or emulsion.Its method of administration Can be oral, percutaneous, vein or intramuscular injection.
Advantage of the invention is that：
Research of the invention by replicating the nsP2 protease to play an important role inhibitor to chikungunya virus, Show tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid to first such as CHIKV Viral nsP2 protease activity has significant inhibition, is expected to turn into the potential medicine of the Alphaviruses such as anti-CHIKV infection Thing.
Brief description of the drawings
Fig. 1 is suppression curve of the tannic acid to CHIKV nsP2 protease.
Fig. 2 is suppression curve of the chloromethoxazole sulfosalicylic acid to CHIKV nsP2 protease.
Fig. 3 is suppression curve of the 2,3- dimercaptosuccinic acids to CHIKV nsP2 protease.
Fig. 4 is suppression curve of the oritavancin diphosphonic acid to CHIKV nsP2 protease.
Embodiment
In order to which the present invention is better described, the embodiment of the present invention is will be described below.
1.CHIKV nsP2 protease expression and purification
According to document (Das, P.K., Merits, A.＆Lulla, A.Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity.J.Biol.Chem.289,5635–5653(2014)),(RAUSALUK,UTTA, QUIRIN T et al..Chikungunya virus infectivity,RNA replication and non- structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue[J].Scientific Reports,2016,6(October):37124.) in method expression with Purify CHIKV nsP2 protease.
Comprise the following steps that：
(1) by the pET28a carrier Transformed E scherichia coli containing coding CHIKV nsP2 protease genes BL21 (DE3) bacterial strain, and with LB plating mediums (kanamycins containing 50mg/L) screening positive clone.
(2) being transferred to 0.8L LB culture mediums after picking positive colony overnight incubation on flat board, (that is mould for card containing 50mg/L Element), as its light absorption value (that is, OD at 600nm wavelength600) when reaching 0.6-0.8, add 0.25mM IPTG (isopropyl sulphur For galactoside, Isopropyl β-D-Thiogalactoside) cultivated 16 hours at 16 DEG C.
(3) height crushes bacterium after collecting cell with 5000rpm centrifugations 10min；Broken bacterium solution is received after centrifuging 30min with 10000rpm Collect supernatant.
(4) supernatant is added into broken bacterium buffer (50mM Na2HPO4, 300mM NaCl, 5% glycerine) pre-equilibration Ni- In NTA affinity columns, destination protein is fully combined with Ni, destination protein is fully enriched with.
(5) uncombined foreign protein, then the broken bacterium buffer with the imidazoles containing 20mM are washed off with broken bacterium buffer (buffer solution) Wash the foreign protein of combination off, when Coomassie brilliant blue G250 detection efflux is constant blue, the most of foreign protein for illustrating to combine is rushed Wash clean.CHIKV nsP2 protease are eluted with the broken bacterium buffer of the imidazoles containing 300mM, it is then dense with 30kD concentration tube Liquid is changed in contracting, is purified with cation-exchange chromatography to obtain the destination protein with electric charge homogeneity.
2.CHIKV nsP2 protease determination of activity
It is more than 95% Dabcyl-DELRLDRAGGYIFSS-Glu (Edans)-NH using purity2(it is purchased from Shanghai gill Biochemical Co., Ltd) it is used as substrate；Dabcyl is that base is quenched for common in 4- (4 '-dimethylaminoazobenzene base) benzoic acid Group, Edans is that 1- amino naphthalenes -8- carboxylic acids are common fluorophor；The instrument of fluorescent strength determining is The wavelength of the multi-functional micropore board detector of M1000Pro all-wave lengths, exciting light and transmitting light is respectively 340nm and 490nm；
Albumen buffer composition is 20mM HEPES (4- hydroxyethyl piperazineethanesulfonic acids), the glycerine of 20% percentage by volume, PH=7.6,2mM dithiothreitol (DTT)；With buffer solution configuration CHIKV nsP2 protease (final concentration 750nM), addition is dissolved in DMSO (dimethyl sulfoxide (DMSO)) compound (final concentration is respectively 50 μM and 20 μM), room temperature place 10min, are rapidly added fluorescence bottom Thing Dabcyl-DELRLDRAGGYIFSS-Glu (Edans)-NH2, concentration of substrate is 30 μM.The record first order fluorescence reading per 6s, 1500s is determined altogether.654rpm shakes 5s, detects fluorescent value.Negative control is not added with alternative sample, other experiment condition all sames.
Using the time as X-axis, fluorescent value is that Y-axis can obtain enzyme activity kinetic curve.The phase of the enzyme reaction recorded by ELIASA Related parameter, according to fluorescence intensity and reaction time, using 500s enzymatic reaction before GraphPad Prism5 software analysis Speed.Set V0For the initial velocity of the enzymatic reaction of without inhibitor, ViFor the initial velocity of the enzymatic reaction of inhibiting.According to Enzyme's reaction speeding, calculate residual activity Ra (Residual Activity, Ra) (V of each compoundi/V0) and suppress Rate Ir (Inhibition Rate, Ir) (1-Vi/V0)。
For residual activity<30% compound carries out secondary screening, excludes the possibility that operational error causes false positive.For surplus Remaining activity<30% compound, design fluorescent quenching experiment.Consider residual activity percentage and the fluorescent quenching of compound Rate it may determine that compound to CHIKV nsP2 protease inhibition.Because the system mainly passes through fluorescence intensity Screening, so, when the compound for having fluorescence compound either similar with Edans all can produce interference to system.In addition, very Structure more similar with Dabcyl can be quenched the fluorescence of system and cause false positive, in order to exclude false positive results, using first general NsP2 protease and fluorogenic substrate reaction a period of time, the system fluorescence of allowing reach maximum Q1, added again in system with it is real The compound of group equivalent is tested, and the fluorescent value size of detection architecture is Q2.Both fluorescent values are calculated according to formula glimmering Optical quenching rate Qr ((Qr=Q1-Q2)/Q2*100%).When fluorescent quenching rate is higher than 20%, it is false positive, as a result needs to exclude； It is positive findings when fluorescent quenching rate is less than 20%.
The present invention relates to the technical field of pharmacy, specifically tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- bis- The application of dimercaptosuccinic acid and oritavancin diphosphonic acid in the infection of the Alphaviruses such as anti-CHIKV, tannic acid, chloromethoxazole sulphur Base salicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid are big to CHIKV nsP2 protease inhibiting rate Ir In 70%, there is very big application potential in terms of the nsP2 protease micromolecular inhibitor of the Alphaviruses such as anti-CHIKV is prepared, It is expected to turn into the potential drug of the Alphaviruses such as anti-CHIKV infection..
Method used above, it is method commonly used in the art unless otherwise specified.
The embodiment of the present invention is these are only, is not intended to limit the invention, it is all in the spiritual and former of the present invention Within then, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Claims (10)

1. tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid are in anti-datum hole Agree the application in refined virus infection.
2. tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- dimercaptosuccinic acids and oritavancin diphosphonic acid are in anti-onychonosus Application in poison infection.
3. application according to claim 1 or 2, wherein, tannic acid, chloromethoxazole sulfosalicylic acid, the mercaptos of 2,3- bis- Base succinic acid and oritavancin diphosphonic acid are chikungunya virus nsP2protease micromolecular inhibitors.
4. application according to claim 1 or 2, wherein, the structural formula of tannic acid is：
5. application according to claim 1 or 2, wherein, the structural formula of chloromethoxazole sulfosalicylic acid is：
6. application according to claim 1 or 2, wherein, the structural formula of DMSA is：
7. application according to claim 1 or 2, wherein, the structural formula of oritavancin diphosphonic acid is：
8. the potential drug of Alphavirus infection is treated or prevented, including：Tannic acid, chloromethoxazole sulfosalicylic acid, 2,3- The one or more and pharmaceutically acceptable carrier and preparation of dimercaptosuccinic acid and oritavancin diphosphonic acid.
9. the potential drug according to claim 8 for treating or preventing Alphavirus infection, wherein, it is described pharmaceutically acceptable Carrier include diluent, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, surfactant, suction The one or more of appendix body, lubricant and synergist.
10. the potential drug for treating or preventing Alphavirus infection according to claims 8, wherein, the potential drug Preparation include injection, tablet, pill, capsule, suspending agent or emulsion.
CN201710908773.3A 2017-09-29 2017-09-29 Using application of the tannic acid as a series of compounds of representative in the infection of the Alphaviruses such as anti-chikungunya Pending CN107823629A (en)

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