CN104398514A - Application of chloroquine to preparing anti-herpesvirus medicines - Google Patents
Application of chloroquine to preparing anti-herpesvirus medicines Download PDFInfo
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- 229960003677 chloroquine Drugs 0.000 title claims abstract description 41
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 title claims abstract description 41
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Classifications
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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
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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- Health & Medical Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of autophagy inhibitors, namely chloroquine, to medicines for treating herpesvirus, in particular to herpesvirus related to Kaposi's sarcoma and EB virus. The chloroquine has the good inhibiting effect on the two kinds of virus; a powerful theoretical basis and a powerful practice basis are provided for further researches and further developments of the anti-herpesvirus medicines; and the important researching and developing values and the important development significance are achieved.
Description
Technical field
The present invention relates to the new opplication of autophagy response inhabitation drug chloroquine in medicine, more specifically, relate to the application of chloroquine to γ-herpesvirus and relevant disease thereof.
Background technology
It is malignant tumor common in HIV sufferers that Kaposi's sarcoma associated herpesvirus KSHV (Kaposi ' s sarcoma-associated herpesvirus) infects the Kaposi's sarcoma KS induced, the AIDS patients of about 20% can with Kaposi's sarcoma, and AIDS-KS patient death rate is high, its 5 years survival rates only have an appointment 8%.In normal population KSHV infect after more than 95% individuality do not show clinical symptoms and ill.But immunosuppressant patient, as in HIV sufferers, Organ Transplantation Patients and chemicotherapy patient, KSHV has very high infection rate and harm greatly, infection can cause Kaposi's sarcoma (Kaposi ' s sarcoma, KS), former effusion lymphoma (primary effusion lymphoma, and the disease such as multicenter Karst slow sick (multicentric castleman disease, MCD) PEL).In other diseases, (as HBV chronic hepatitis, drug abuse and Senile disease) also finds very high KSHV infection rate and malignant tumor in recent years, draws attention just gradually.
Epstein-Barr virus (Epstein-barr virus, EBV) is also a member in herpes virus group, belongs to γ-herpesvirus with KSHV.Current EBV infects the crowd in the whole world more than 90%, with multiple mankind's disease association, comprises infectious monocytosis, hairy leukoplakia disease, aids related lymphoma, and lymphoproliferative disease multiple after solid organ transplantation.In addition, EBV is the virus that can cause cancer found the earliest, relevant to Several Kinds of Malignancy, as Burkitt lymphoma, Hodgkin lymphomas, non-Hodgkin lymphomas and some epithelial tissue cancers (gastric cancer etc. as nasopharyngeal carcinoma, some form), people's showing great attention to for EBV has also been caused as the area occurred frequently of nasopharyngeal carcinoma in Guangzhou.
Do not have special anti-γ-herpesvirus medicament up to now, vaccine is also still in development.
Utilizing autophagy to react treatment γ-herpesvirus-related disease may be a good developing direction, and Rapamycin is the most effective current KS medicine, and this medicine can induce autophagy to react significantly; Although autophagy reaction concrete effect wherein waits to confirm, undeniable autophagy reaction has played critical function wherein.Equally suppress the autophagy response inhabitation albumen vFLIP of encoding viral can the growth of remarkable Tumor suppression by polypeptide.These researchs show that autophagy reaction is very potential KSHV therapy target.EBV and KSHV homology is very high, RTA is as the total burst times of KSHV and EBV and early protein, and be virus is transformed into burst times smoothly key factor from incubation period, RTA can promote that autophagy is reacted, and once suppress autophagy reaction, will be suppressed from incubation period to the conversion of burst times.
Summary of the invention
The invention provides the application of autophagy reaction suppressor chloroquine in anti-gamma herpes viruses medicine.
The structural formula of described chloroquine is such as formula shown in I:
Described gamma herpes viruses is Kaposi's sarcoma associated herpesvirus or Epstein-Barr virus.
Chloroquine (chloroquine, CQ) suppresses medicine as autophagy, and it suppresses the principle of autophagy to be to change lysosomal pH, thus suppresses the fusion of lysosome and autophagosome, affects the process of autophagy further.In addition, chloroquine can interact with DNA, suppresses copying and transcribing of some gene.Find that it has the effect of anti-KSHV by a series of research, the expression of KSHV burst times albumen can be suppressed, reduce in cell and extracellular viral yield, possible mechanism is that chloroquine is by suppressing autophagy reaction thus suppressing the cracking of virus to copy, another possibility is exactly that chloroquine can the promoter of specific binding RTA, thus suppresses cracking to copy.For the suppression of EBV, chloroquine only has inhibitory action to extracellular viral yield, and this may be the lysosomal pH change that chloroquine causes, and autophagosome is accumulated, and suppresses the effect of the assembling release of virus.In the file applied for before, do not relate to the research about chloroquine treatment KSHV and EBV relevant disease and application.This researches and develops for further antiviral drugs and provides strong theoretical basis and practical basis, has important research and development value and development significance.
The present invention has the following advantages:
(1) in the cell after experiment finds chloroquine process, the expression of KSHV burst times albumen obviously reduces, and the virion content of intraor extracellular also has obvious decline, and EBV also has obvious decline at extracellular content.
(2) these inhibitory action are concentration dependent, and just have good antivirus action in lower concentration.
(3) chloroquine toxicity is lower, in ready-made no cytotoxicity phenomenon in HEK-293 cell.
Accompanying drawing explanation
Fig. 1: chloroquine in BCBL1 cell on the impact of the expression of KSHV albumen incubation period LANA, burst times albumen RTA, K8, ORF45, ORF64.
Fig. 2: chloroquine in iSLK-219 cell to KSHV associated protein RTA, LANA2, ORF45, K8 express effect.
Fig. 3: chloroquine is on the impact of the extracellular KSHV viral yield of BCBL1.
Fig. 4: chloroquine is on the impact of BCBL1 intracellular KSHV virion quantity.
Fig. 5: chloroquine is on the impact of the extracellular EBV viral yield of HR-1.
Fig. 6: chloroquine is on the impact of HR-1 intracellular EBV virion quantity.
Detailed description of the invention
The present invention is further described below in conjunction with specific embodiment.Unless stated otherwise, the present invention adopts reagent, equipment and method are conventional commercial reagent, equipment and the conventional method used of the art.
KSHV, EBV are as γ-herpesvirus, and biocycle is all divided into incubation period and burst times, enter incubation period rapidly after cell is infected through of short duration acute reaction, and now burst times gene is not expressed; And when host immune suppresses, through special stimulation, cell enters burst times, virus amplification is bred, burst times gene expression.Therefore, the correlative protein expression detecting incubation period and burst times can be used as a kind of means of assessment medicine for virus function.
Embodiment 1
1, get well-grown BCBL1 cell, be inoculated in 6 hole clear flat bottom plates, every hole 2 × 10
6cell, is divided into induction group and non-induced group by cell.The culture medium used is complete medium: DMEM in high glucose, 10% hyclone and 1% dual anti-, condition of culture be 5% carbon dioxide, 37 DEG C;
2, after 3h, induction group adds derivant TPA, and final concentration is 20ng/mL;
3, add chloroquine respectively after inducing 3h, final concentration is respectively 0 μM, 5 μMs, 10 μMs, 15 μMs, 20 μMs;
4,48h is cultivated after dosing, 1000rpm, 10min harvesting;
5, carry out protein blot experiment, result such as Fig. 1 shows: in BCBL1 cell, the expression of KSHV associated protein RTA, ORF45, K8, ORF64 is subject to the suppression of chloroquine effect after induction 3h, and inhibitory action is certain concentration dependent.This illustrates that chloroquine have impact on the albumen synthesis of KSHV at the virolysis initial stage of copying.
Embodiment 2
1, get well-grown iSLK-219 cell, 1:4 is inoculated in 6 hole clear flat bottom plates, cell is divided into induction group and non-induced group.
2,12h cell attachment, induction group adds derivant Dox and NaB, and final concentration is respectively 1 μ g/mL and 0.3 μM;
3, add chloroquine after inducing 3h, final concentration is respectively 0 μM, 2 μMs, 5 μMs, 8 μMs, 10 μMs, 15 μMs, 20 μMs, 25 μMs;
4, after dosing 48h, with cell scraper plate cell scraped from plate and take out, cold PBS rinsing, 1000rpm, 10min, 4 DEG C of centrifugal collecting cells;
5, carry out protein blot experiment, experimental result as shown in Figure 2: in iSLK-219 cell, the expression of KSHV associated protein RTA, ORF45, K8 is by the suppression of chloroquine effect, and inhibitory action is certain concentration dependent.
Embodiment 3
1, get well-grown cell line BCBL1, plant in 48 orifice plates, cell consumption is 1 × 10
5/ hole, is divided into cell and does not induce group (3 hole) and induction group (15 hole);
2, after 3h, induction group adds derivant TPA, and final concentration is 20ng/mL;
3, the chloroquine of respective concentration is added after inducing 3h, concentration 0 μM, 5 μMs, 10 μMs, 15 μMs, 20 μMs respectively, each concentration 3 holes;
4, harvesting after 96h, 10000g, gets supernatant for 4 DEG C;
5, extract extracellular virus DNA, method is as follows:
A. get 200 μ L supernatant, add 2 μ L DNase I, 20 μ L10 × DNase I buffer, hatch 1h in 37 DEG C of incubators;
B. in aforesaid liquid, 0.5M EDTA 10 μ L is added, mixing, cessation reaction;
C.80 DEG C water-bath deactivation 10min;
D. add 20 μ L proteinase K solution, and then add 200 μ L AL buffer, vortex mixes;
E.56 DEG C water-bath 10min;
F. add DNA in 440 μ L phenol chloroform virions, vortex mixes;
G.12000rpm, 4 DEG C of centrifugal 10min;
H. centrifugal rear layering, gets supernatant about 300 μ L, in another ep pipe.Avoid the phenol chloroform being extracted into lower floor;
I. add 5M NaCl solution 6 μ L, add dehydrated alcohol 750 μ L, finally add glycogen 1 μ L;
J. put into-80 DEG C of refrigerators after fully mixing, place 1h;
K. sample is taken out from refrigerator, 12000rpm, 4 DEG C, the centrifugal rear removal supernatant of 30min;
L.Ep pipe inner bottom part occurs that DNA precipitates, and washes, closes the lid, turn upside down several times with 1mL70% ice ethanol;
M.12000rpm, 4 DEG C of centrifugal 10min, abandon supernatant;
N.12000rpm, 4 DEG C of centrifugal 10min, the ethanol that removing is remaining;
O. uncap, allows unnecessary ethanol volatilize;
P.3min 40 μ L ddH2O dissolving DNAs are added afterwards
6, Real-time quantitative PCR detects viral level, and result as shown in Figure 3.Result show, cracking copy early stage chloroquine for KSHV the extracellular output of BCBL1 be concentration dependent suppression.
Embodiment 4
1, get well-grown cell line BCBL1, be inoculated in six orifice plates, every hole 2 × 10
6cell, is divided into induction group and non-induced group by cell;
2, after 3h, induction group adds derivant TPA final concentration to 20ng/mL;
3, add chloroquine after inducing 3h, final concentration is respectively 0 μM, 5 μMs, 10 μMs, 15 μMs;
4, harvesting after 48h, extracts intracellular DNA;
5, Real-time quantitative PCR detects the copy number of intracellular virus, and result as shown in Figure 4.
Result show, chloroquine for KSHV the intracellular content of BCBL1 be concentration dependent suppression.
Embodiment 5
1, get well-grown cell line HR-1, plant in 48 orifice plates, cell consumption is 1 × 10
5/ hole, is divided into cell and does not induce group (3 hole) and induction group (15 hole);
2, after 3h, induction group adds derivant TPA and NaB, and final concentration is respectively 20ng/mL and 3 μM;
3, add the chloroquine of respective concentration after inducing 3h, concentration is 0 μM, 10 μMs, 25 μMs, 50 μMs each concentration, 3 multiple holes respectively;
4, harvesting after 96h, centrifugal 10 minutes of 10000g, gets supernatant for 4 DEG C;
5, extract extracellular virus DNA, method is as follows:
A. get 200 μ L supernatant, add 2 μ L DNase I, 20 μ L10 × DNase I buffer, hatch 1h in 37 DEG C of incubators;
B. in aforesaid liquid, 0.5M EDTA 10 μ L is added, mixing, cessation reaction;
C.80 DEG C water-bath deactivation 10min;
D. add 20 μ L proteinase K solution, and then add 200 μ L AL buffer, vortex mixes, 56 DEG C of water-bath 10min;
E. add DNA in 440 μ L phenol chloroform virions, vortex mixes;
F.12000rpm, 4 DEG C of centrifugal 10min;
H. centrifugal rear layering, gets supernatant about 300 μ L, in another ep pipe.Avoid the phenol chloroform being extracted into lower floor;
I. add 5M NaCl solution 6 μ L, add dehydrated alcohol 750 μ L, finally add glycogen 1 μ L;
G. put into-80 DEG C of refrigerators after fully mixing, place 1h;
K. sample is taken out from refrigerator, 12000rpm, 4 DEG C, the centrifugal rear removal supernatant of 30min;
L.Ep pipe inner bottom part occurs that DNA precipitates, and washes, closes the lid, turn upside down several times with 1mL70% ice ethanol;
M.12000rpm, 4 DEG C of centrifugal 10min, abandon supernatant;
N.12000rpm, 4 DEG C of centrifugal 10min, the ethanol that removing is remaining;
O. uncap, allows unnecessary ethanol volatilize;
P.3min 40 μ L ddH2O dissolving DNAs are added afterwards
6, Real-time quantitative PCR detects viral level, and result such as Fig. 5 shows, and chloroquine has inhibitory action to the extracellular EBV viral yield of HR-1, and suppresses in concentration dependent.
Embodiment 6
1, get well-grown cell line HR-1, be inoculated in six orifice plates, every hole 2 × 10
6cell, is divided into induction group and non-induced group by cell;
2, after 3h, induction group adds derivant TPA, NaB, final concentration be respectively 20ng/mL, 3 μMs;
3, after induction, 3h adds chloroquine, and final concentration is respectively 0 μM, 10 μMs, 25 μMs, 50 μMs;
4, harvesting after 48h, extracts intracellular DNA;
5, Real-t ime quantitative PCR detects the copy number of intracellular virus, and result as shown in Figure 6.
Experimentally result can find out that chloroquine has no significant effect at intracellular content EBV.
Embodiment 7
MTS (3-(4,5-dimethylthiazol-2-yl)-5 (3-carboymethoyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt) be a kind of tetrazole compound of new synthesis, it is identical with the application principle of MTT, namely be reduced into first a ceremonial jade-ladle, used in libation product coloured separately by the multiple dehydrogenase in living cells mitochondrion, the viable count of its shade and some sensitive cells strain is within the specific limits in height correlation.According to the absorbance (OD value) of the 490nm recorded, judge living cells quantity, OD value is larger, and cytoactive is stronger, then represent that drug toxicity is less.
1, inoculating cell, is made into individual cells suspension with the DMEM culture fluid containing 10% tire calf serum by HEK-293 cell, is inoculated into 96 orifice plates, every pore volume 200ul with 1000, every hole cell;
2,24h adherent after add chloroquine, final concentration is respectively 40 μMs, 20 μMs, 15 μMs, 10 μMs, 5 μMs, 0 μM;
3, after cultivating 48h, every hole adds MTS solution 20ul, continues to hatch 2 ~ 4h in incubator;
4, select 490nm wavelength, enzyme linked immunological monitor measures each hole absorbance value, observe compound to the cytotoxicity of 293 cells.Experimental result is as shown in table 1, CC50>>40 μM, far below the valid density suppressing virus replication, shows chloroquine in normal cell without obvious cytotoxicity.
Table 1 cytotoxicity experiment
Claims (4)
1. the application of chloroquine in the anti-γ-herpesvirus medicament of preparation.
2. apply as claimed in claim 1, it is characterized in that, described chloroquine is such as formula shown in (1).
3. apply as claimed in claim 1, it is characterized in that, described herpesvirus is Kaposi's sarcoma associated herpesvirus.
4. apply as claimed in claim 1, it is characterized in that, described herpesvirus is Epstein-Barr virus.
Priority Applications (1)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1083064A (en) * | 1992-04-17 | 1994-03-02 | 史密丝克莱恩比彻姆公司 | Indolizino [1, the 2-b] quinolinones that replaces |
| WO2002017917A1 (en) * | 2000-08-29 | 2002-03-07 | The University Of Mississippi | Manzamines for treatment of drug resistant infection |
| WO2007059372A2 (en) * | 2005-11-09 | 2007-05-24 | St. Jude Children's Research Hospital | Use of chloroquine to treat metabolic syndrome |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1083064A (en) * | 1992-04-17 | 1994-03-02 | 史密丝克莱恩比彻姆公司 | Indolizino [1, the 2-b] quinolinones that replaces |
| WO2002017917A1 (en) * | 2000-08-29 | 2002-03-07 | The University Of Mississippi | Manzamines for treatment of drug resistant infection |
| WO2007059372A2 (en) * | 2005-11-09 | 2007-05-24 | St. Jude Children's Research Hospital | Use of chloroquine to treat metabolic syndrome |
Non-Patent Citations (1)
| Title |
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| NANCIMAE MILLER等: "Epstein-Barr Virus Enters B Cells and Epithelial Cells by Different Routes", 《JOURNAL OF VIROLOGY》 * |
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