CN105832722B - Compound PACMA31 is preparing the application in anti-infection of hepatitis C virus drug - Google Patents
Compound PACMA31 is preparing the application in anti-infection of hepatitis C virus drug Download PDFInfo
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- CN105832722B CN105832722B CN201610245898.8A CN201610245898A CN105832722B CN 105832722 B CN105832722 B CN 105832722B CN 201610245898 A CN201610245898 A CN 201610245898A CN 105832722 B CN105832722 B CN 105832722B
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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
- A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
- A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
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Abstract
The invention discloses a kind of compound PACMA31 to prepare the application in anti-infection of hepatitis C virus drug.The anti-infection of hepatitis C virus drug is that PACMA31 as sole active agent or includes the pharmaceutical composition of PACMA31.The present invention experiments prove that, PACMA31 can inhibit the infection of Hepatitis C Virus in rna level and protein level, can significantly inhibit HCV invasion target cell.The present invention provides a kind of purposes for preparing anti-hepatitis c virus infection medicine for PACMA31.The present invention provides new source to find anti hepatitis C virus drug.
Description
Technical field
The present invention relates to a kind of purposes of compound, specifically, being that compound PACMA31 is preparing anti-hepatitis C virus sense
Contaminate the application in drug.
Background technique
HCV (Hepatitis C virus, HCV) is single positive chain RNA virus, belongs to flaviviridae Hepacivirus,
It is the important pathogen body for causing virus hepatitis mainly through blood born.The chronic rate of HCV infection is more than the chronic sense of 80%, HCV
Dye can cause cirrhosis and hepatocellular carcinoma, it was reported that the year of medical history 20 years or more patients with chronic hepatitis C, cirrhosis occurs
Rate is 10-15%, and is every year liver cancer there are about the development of the liver cirrhosis patient of 1-7%.It is treatment third that interferon, which combines Ribavirin,
The major measure of type hepatitis, however the pharmaceutical composition is only effective to the HCV infection person of 50-70%, but also there are costly, secondary
Act on big, patient dependence difference disadvantage.Although some pharmaceutical companies develop direct antiviral drugs in succession in recent years
(direct-acting antiviral agents, DAAs) promotes HCV-Ab IgG to a certain extent and treats, however these medicines
Object price is universal higher, and patient's financial burden has been further aggravated.Thus still need to seek different therapeutic agent and means.
PACMA31, full name propynoic acid carbamoyl methyl amide, also known as N- (2,4-
Dimethoxyphenyl)-N-(1-oxo-2-propyn-1-yl)-2-(2-thienyl)glycyl-glycine ethyl
Ester, chemical formula C21H22N2O6S, the CAS number of logging in 1401089-31-3, chemical structural formula are as follows:
PACMA31 is a kind of amides compound, and the researcher from University of Southern California has found that PACMA31 is a kind of cell
Toxic agent has obviously toxicity to ovarian cancer cell.Its mechanism of action is that PACMA31 can selectively inhibit disulfide bond different
The activity of structure enzyme (Protein disulfide isomerase, PDI).PDI is highly expressed in oophoroma, PACMA31 with
PDI is target spot, intervenes the folding of protein, after occurring the albumen of many false foldings in cancer cell, will cause cell pressure
Power eventually leads to cancer cell death.
Researcher also found PDI participate in human immunodeficiency virus (human immunodeficiency virus,
HIV the film fusion process of target cell) is invaded, PDI can promote poisoning intrusion target cell.HIV passes through memebrane protein gp120 and cell
The CD4 of film is combined, and the conformation of memebrane protein gp120 and gp41 change under the action of PDI, and then viral endocytosis enters cell
And releasing virus genome is merged by film.The active small molecule compound of PDI or PDI antibody is inhibited to be able to suppress inhibition of HIV
Invasion and propagation, significantly inhibit virus infection level.
HCV also passes through receptor and merges in conjunction with film as tunicary single positive chain RNA virus when invading liver cell, this
It is closely similar when one feature is with HIV infection cell.And there is also disulfide bond on the envelope protein E1 E2 of HCV, and finger has been reported
The disulfide bond of E2 is very important the invasion of virus out.Therefore protein disulfide isomerase (PDI) is thin in HCV invasion
Important function may also be participated in during born of the same parents, however have not yet to see PDI and HCV interaction research, also not about
PDI inhibitor PACMA31 document report relevant to Hepatitis C Virus.
Summary of the invention
The purpose of the present invention is to provide PACMA31 to prepare the application in anti-hepatitis c virus infection medicine.This hair
The bright PACMA31 is amides compound, and chemical formula C21H22N2O6S, No. CAS is 1401089-31-3, chemical structural formula
It is as follows
PACMA31 is embodied in the application prepared in anti-hepatitis c virus infection medicine in the present invention are as follows: the anti-hepatitis
Virus infective medicament is that PACMA31 as sole active agent or includes the pharmaceutical composition of PACMA31.
It include PACMA31 and other pharmaceutically acceptable auxiliary materials in aforementioned pharmaceutical compositions.Wherein, the pharmaceutical preparation
It can be ejection preparation or oral preparation, the ejection preparation can be freeze drying powder injection, and the oral preparation can be discrete piece
Agent, capsule or granule.
The present invention experiments prove that, PACMA31 can inhibit the sense of Hepatitis C Virus in rna level and protein level
Dye can significantly inhibit HCV invasion target cell.
The present invention has the advantages that the present invention provides a kind of anti-hepatitis c virus infection medicine of preparing for PACMA31
Purposes.The present invention provides new source to find anti hepatitis C virus drug.
Detailed description of the invention
Attached drawing 1 is the experimental result picture of PACMA31 dose-dependent inhibition hepatitis c viral replication, and wherein A is
Cytotoxicity (CC50=200nM) of the PACMA31 to Huh7 cell；B is that PACMA31 inhibits HCV duplication, medium effective concentration
For 70nM.
Attached drawing 2 is that PACMA31 concentration-time depended inhibits to continue the experimental result picture of HCV infection.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with specific embodiment.
Embodiment 1
The present embodiment is the test that PACMA31 inhibits hepatitis pseudovirus (HCVpp) infection.Facilitated using pseudovirus mode
It more safely tests, harm reduction.
Trial drug, reagent and material are as follows:
1. compound: PACMA31
2. cell line Huh7: human hepatoma cell strain is (referring in particular to following article: Liu Y, Zou Z, Zhu B, et
al.CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of
Hepatitis C Virus(HCV)Infection.Int J Mol Sci.2013 Dec 13；14(12):24230-41.)
3. cell line 293T: human embryonic kidney cell line is (referring in particular to following article: Liu Y, Zou Z, Zhu B, et
al.CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of
Hepatitis C Virus(HCV)Infection.Int J Mol Sci.2013 Dec 13；14(12):24230-41.)
4. cell culture fluid: it prepares, it is green containing 10% fetal calf serum, 0.03% glutamine, nonessential amino acid, ammonia benzyl
Mycin and streptomysin 100U/ml adjust pH to 7.4.
5. cell dissociation buffer: preparing, contain 0.25% trypsase, use phosphate buffered saline.
7.HCVpp:HCV pseudovirus is (referring in particular to following article: Wang W, Guan M, Liu Y, et al.Alanine
scanning mutagenesis of hepatitis C virus E2 cysteine residues:Insights into
E2 biogenesis and antigenicity.Virology.2014 Jan 5；448:229-37.)
Experimental method includes the following steps:
(1) preparation of HCVpp and infection experiment
1. the HEK293T cell communicating in logarithmic growth phase is inoculated in 24 orifice plates, it is placed in 37 DEG C of 5%CO2Incubator
Interior overnight incubation.
2. observing cell growth condition, when cell grows to 70% and converges, plasmid transfection is carried out.By liposome 2ul with
And mouse leukemia virus gag plasmid, reporter gene GFP expression plasmid and HCV envelope protein E1 E2 expression plasmid are placed in
In 100ul opti-MEM culture solution, mixes, be placed at room temperature for 20min.
3. absorbing HEK293T cells and supernatant, 400ul opti-MEM culture solution is added, adds liposome-plasmid
Tissue culture plate is placed in incubator and cultivates by mixed liquor.
Cells and supernatant is absorbed after 4.6h, and the DMEM culture medium that 500ul contains 10% fetal calf serum is added, is placed in incubator
48h is cultivated, cells and supernatant is collected, high speed centrifugation removes cell fragment.
5. culture supernatant of the 50ul containing HCVpp is added in every hole after adherent by Huh7 cell inoculation in 96 orifice plates, changed after 6h
The fresh DMEM culture medium for containing 10% fetal calf serum continues to cultivate 72h, counts gfp positive cell under fluorescence microscope
Number.
(2) Inhibition test that PACMA31 infects HCVpp
The Huh7 cell of logarithmic growth phase is inoculated with 96 orifice plates, inoculum density 1 × 104Additionization afterwards is cultivated for 24 hours in a/hole
Object and HCVpp are closed, compound removes compound and HCVpp mixed liquor after cultivating 6h by the concentration dilution of 10,25,50,100nM,
Replacement culture medium continues to cultivate, and reads each hole gfp positive cell number after 72h under immunofluorescence microscopy.
The result of Inhibition test is as shown in the table:
Inhibitory activity of 1 PACMA31 of table to hepatitis pseudovirus (HCVpp)
| PACMA31 concentration (nM) | 10 | 25 | 50 | 100 |
| Inhibiting rate (%) | 4.6 | 23.7 | 36.5 | 47.8 |
Embodiment 2
The present embodiment is the test that various dose PACMA31 inhibits hepatitis C virus (HCVcc) infection
The trial drug, reagent and material of the present embodiment are the same as embodiment 1
Experimental method is as follows:
(1) preparation of HCVcc
1. virus amplification
J6JFH1 mosaic type HCVcc (105Ffu/ml), 50ul infection is taken to be inoculated in the huh7.5.1 cell of 24 orifice plates, it is secondary
Day changes liquid, then according to cell density secondary culture, cell growth state is observed, to cell caused by viral fast breeding
After pathological effect (CPE) occurs, culture supernatant is collected, 12000rpm is centrifuged 5min, discards cell fragment, 0.45 μm of filter membrane mistake
Filter takes and measures in right amount for virus titer, freezes after remaining packing spare in -80 DEG C of refrigerators.
2. titration of virus
24 orifice plate of Huh7 cell inoculation, density 5 × 104A/hole after cultivating 12h, discards culture supernatant, and every hole is added
The HCVcc of 10 times of gradient dilutions of 100ul, is incubated for 6h altogether, is changed to fresh culture medium, continues to cultivate 72h, glimmering by being immunized
Light method detects HCV positive cell, and for primary antibody with the diluted HCV antibody positive patients serum of 1:100, secondary antibody is diluted with 1:200
The goat anti-human igg of FITC label.In fluorescence microscopy microscopic observation luminescent cell, and records the last one and green fluorescence can be observed
Fluorescencepositive cell number and corresponding dilution gradient in the hole of positive cell calculate focus forming unit/ml (ffu/
Ml) numerical value represents HCVcc titre with this.
Cytotoxicity of the 3.PACMA31 to Huh7 cell
In order to evaluate PACMA31 to the cytotoxicity of Huh7 cell, by 96 orifice plate of Huh7 cell inoculation, inoculum density 5
×104The PACMA31 (0,10,25,50,100nM) of various dose is added in a/hole after culture 6 hours, after being incubated for 6 hours altogether,
Cell viability is detected using CCK8 cell viability detection kit, calculates the drug dose (median of half cytotoxicity
Cytotoxic concentration, CC50).
The experiment of 4.PACMA31 inhibition HCV infection
The Huh7 cell in logarithmic growth phase is taken, adjustment cell concentration is 1 × 105A/ml takes 100 μ l to be inoculated with 96 holes
Compound and HCVcc is added in plate, culture afterwards for 24 hours, and compound presses 0,10,25,50,100nM concentration dilution, and 37 degree are cultivated 6 hours
Compound and HCVcc mixed liquor are removed afterwards, are changed culture solution and are continued to cultivate；Real-time quantitative PCR and western are carried out after 48 hours
Blot method detects the intracellular Hepatitis C virus RNA of Huh7 and protein level, calculates medium effective concentration (concentration
For 50%of maximal effect, EC50).
It is tested by cellulotoxic experiment and HCV infection, it has been found that compound PACMA31 dose-dependent inhibition HCVcc
Duplication (as shown in Fig. 1), EC50 70nM, CC50 are about 200nM.
Embodiment 3
The present embodiment is that PACMA31 concentration-time depended inhibits to continue the experiment of HCV infection
The trial drug, reagent and material of the present embodiment are the same as embodiment 1.
Experimental method is as follows
1, HCVcc persistent infection Huh7 cell construction
Huh7 cell inoculation 10cm culture dish, inoculum density 5 × 106It is infected after culture 12 hours with HCVcc in a/hole
(MOI=2), in the DMEM culture medium containing 10% fetal calf serum, culture to cell is paved with plate, with 1 to 3 passage, continues
Same CMC model.Continuous culture 14 days, that is, establish the Huh7 cell strain of HCV persistent infection.
2, PACMA31 tests the HIV suppression of duration HCVcc infection cell
By 96 orifice plate of Huh7 cell inoculation of HCVcc persistent infection, density 5 × 104A/hole, in CO2Culture 6 is small in incubator
Shi Hou is added compound PACMA31 (70nM, 100nM), continues culture 6 hours, washs cell 3 with 100 μ l complete mediums
It is secondary, fresh culture is added, continues to cultivate, and the different time points (1 day, 2 days, 3 days, 5 days, 9 days) after compound addition, receives
Collect cell, extracts cell total rna, the rna level of intracellular HCV is detected with real time quantitative PCR method.
Three, experimental result
As shown in Fig. 2, PACMA31, which infects duration HCVcc, is presented time-dose dependence inhibiting effect.In drug
When acting on the 9th day, the rna level of intracellular HCV reduces by 60% or more.
It is above-mentioned the experimental results showed that, compound PACMA31 can significant antihepatitis C virus activity, therefore can be used for
Prepare the drug of anti-hepatitis c virus.The present invention provides new source to find anti hepatitis C virus drug.
Above embodiments are only the preferred embodiment of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, all other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.Meanwhile the above embodiments are only used to help understand method and its core of the invention
Thought is thought, for those of ordinary skill in the art, according to the thought of the present invention, in specific embodiments and applications
It will change, in conclusion the contents of this specification are not to be construed as limiting the invention.
Claims (3)
1. compound PACMA31 is amides chemical combination preparing the application in anti-infection of hepatitis C virus drug, the PACMA31
Object, chemical formula C21H22N2O6S, No. CAS is 1401089-31-3, and chemical structural formula is as follows
It is characterized by: the anti-infection of hepatitis C virus drug is PACMA31 as sole active agent.
2. compound PACMA31 is amides chemical combination preparing the application in anti-infection of hepatitis C virus drug, the PACMA31
Object, chemical formula C21H22N2O6S, No. CAS is 1401089-31-3, and chemical structural formula is as follows
It is characterized by: the anti-infection of hepatitis C virus drug is the pharmaceutical composition comprising PACMA31.
3. compound PACMA31 according to claim 2 is preparing the application in anti-infection of hepatitis C virus drug, feature
It is: includes PACMA31 and other pharmaceutically acceptable auxiliary materials in the pharmaceutical composition, the pharmaceutical preparation is injection
Preparation or oral preparation, the ejection preparation are freeze drying powder injection, and the oral preparation is discrete piece agent, capsule or granule.
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| CN201610245898.8A CN105832722B (en) | 2016-04-20 | 2016-04-20 | Compound PACMA31 is preparing the application in anti-infection of hepatitis C virus drug |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012129452A2 (en) * | 2011-03-22 | 2012-09-27 | University Of Southern California | Propynoic acid carbamoyl methyl-almides and pharmaceutical compositions and methods based thereon |
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| CN104388430B (en) * | 2014-10-30 | 2019-06-07 | 昆明理工大学 | Target siRNA and its application of people's protein disulfide bond isomerase gene |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012129452A2 (en) * | 2011-03-22 | 2012-09-27 | University Of Southern California | Propynoic acid carbamoyl methyl-almides and pharmaceutical compositions and methods based thereon |
Non-Patent Citations (2)
| Title |
|---|
| Broad-spectrum antivirals against viral fusion;Frederic Vigant等;《NATURE REVIEWS MICROBIOLOGY》;20150731;第13卷;426-437 * |
| 新药PACMA可治疗卵巢癌;不详;《中国处方药》;20121231;第10卷(第5期);38 * |
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