CN105617381B - The new application of histon deacetylase (HDAC) inhibitor treatment β subfamily herpesviral - Google Patents
The new application of histon deacetylase (HDAC) inhibitor treatment β subfamily herpesviral Download PDFInfo
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Abstract
The invention discloses the new applications of histon deacetylase (HDAC) inhibitor treatment β subfamily herpesviral.Specifically, the present invention provides the purposes of a kind of histon deacetylase (HDAC) (HDAC) inhibitor or its pharmaceutically acceptable salt, are used to prepare the pharmaceutical composition for the treatment of β subfamily herpesviral.It is demonstrated experimentally that the hdac inhibitor of low concentration there can be efficient inhibiting rate to β subfamily herpesvirus infection, inhibit efficiency up to 99.69-99.97%.Therefore, the active drug using hdac inhibitor as clinical treatment β subfamily herpesvirus infection.
Description
Technical field
The present invention relates to viral therapy fields.β subfamily is treated in particular it relates to a kind of histon deacetylase (HDAC) inhibitor
The new application of herpesviral.
Background technique
Herpesviral (Herpesviruses) is the medium sized distrand DNA virus of a group, is divided into according to its physicochemical property
Tri- subfamilies of α, β, γ.α herpesviral (such as herpes simplex virus, one herpes zoster virus of varicella) growth rate is fast, causes thin
Born of the same parents' lesion.β herpesviral (such as cytomegalovirus), growth cycle is long, and infection cell forms giant cell.Gamma herpes viruses (such as EB
Virus), the target cell of infection is lymphoid cell, can cause lymphocytic hyperplasia.The host range of herpesvirus infection is extensive, can feel
Contaminate the mankind and other vertebrates.Cause mankind's generation has 7 kinds of herpesvirals.
Human cytomegalovirus (HCMV) belongs to the β subfamily of herpes virus group, is officially named giant cell pneumonia virus.
Cytomegalovirus infection is extremely extensive in China, and general population HCMV antibody positive rate is 86%~96%, and pregnant woman is 95% left
The right side, infant are 60%~80%.The main latency site of HCMV is vascular endothelial cell and vascular smooth muscle cells, is furthermore also dived
It lies prostrate in lymphocyte, monocyte, polymorphonuclear leukocyte, epithelial cell and fibroblast.Cytomegalovirus is for being immunized function
Can normal people do not have the apparent pathogenic or even overwhelming majority simultaneously and show as symptomless infection, still, for pathologic or
The low crowd of physiologic immune, such as AIDS patient, organ or bone marrow transplant patient, developmental character immune deficiency fetus and new
Raw youngster etc. is the Important cause of disease for causing serious conditions or increasing the death rate.Cytomegalovirus infection is to thymus development and spleen
Cell, mononuclear phagocytic cells, the function of NK cell and CTL cell have significant impact, and the immune function of body can be caused to drop
It is low, especially cellular immune function decreased.Human cytomegalovirus is most common, the maximum a kind of virus of harm in intrauterine infection,
And it there is no effective treatment method so far.
Therefore, there is an urgent need in the art to develop one kind can effectively treat β subfamily herpesviral (especially human cytomegalovirus disease
Poison) drug.
Summary of the invention
The present invention provides a kind of new applications of histon deacetylase (HDAC) inhibitor, are used for β subfamily herpesviral
Treatment.
First aspect present invention provides a kind of histon deacetylase (HDAC) (histone deacetylase, HDAC) suppression
The purposes of preparation or its pharmaceutically acceptable salt is used to prepare the pharmaceutical composition for the treatment of β subfamily herpesviral.
In another preferred example, the hdac inhibitor is also used to inhibit β subfamily herpes virus replication, assembling.
In another preferred example, the hdac inhibitor include Vorinostat (Vorinostat,
SAHA), 8- hydroxyl -5- nitroquinoline (NSC-3852), 4- acetylaminohydroxyphenylarsonic acid N- (2'- aminophenyl)-benzamide (CI-
994), N- hydroxyl -7- [5- (4- three-level butoxy glycosyl aminobenzene) -3- isoxazole formamide] heptamide (BML-281).
In another preferred example, the β subfamily herpesviral includes human cytomegalovirus (CMV), human herpes virus 6
Type.
In another preferred example, the β subfamily herpesviral is human cytomegalovirus.
In another preferred example, the hdac inhibitor is Vorinostat.
In another preferred example, the pharmaceutical composition includes the hdac inhibitor of safe and effective amount, and pharmaceutically may be used
The carrier of receiving.
In another preferred example, anti-β subfamily herpesvirus medicament is also contained in the pharmaceutical composition.
In another preferred example, the anti-β subfamily herpesvirus medicament include Ganciclovir, acyclovir, interferon,
Interleukin.
In another preferred example, the pharmaceutical composition contains 0.1-100 μM, and preferably 1-50 μM, be 2- more preferably
10 μM of hdac inhibitor or its pharmaceutically acceptable salt.
In another preferred example, the drug is applied to mammal in the following manner: oral, intravenous injection, office
Portion's injection.
In another preferred example, the dosage form of the pharmaceutical composition is peroral dosage form.
Second aspect of the present invention provides a kind of method of the inhibition β subfamily herpesviral of external non-therapeutic, in HDAC
In the presence of inhibitor, cultivates β subfamily herpesviral or infect the cell of β subfamily herpesviral.
In another preferred example, the effective quantity of the hdac inhibitor is 1-100 μM, preferably, being 5-20 μM, more preferably
Ground is 2-10 μM.
In another preferred example, the inhibition β subfamily herpesviral include inhibit β subfamily herpesviral duplication and/
Or assembling.
In another preferred example, the cell includes human fibroblasts (HF), human embryonic lung diploid fibroblast
(MRC5), Human RPE Cells in Vitro (ARPE19).
Third aspect present invention provides a kind of for inhibiting the pharmaceutical composition of β subfamily herpesviral, the drug
Composition includes hdac inhibitor or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
Fourth aspect present invention provides a kind of method for treating β subfamily herpesvirus infection, to object in need for the treatment of
The hdac inhibitor or its pharmaceutically acceptable salt for applying safe and effective amount, to treat β subfamily herpesvirus infection.
In another preferred example, the object in need for the treatment of is mammal, preferably, being people, mouse, rat.
In another preferred example, the application includes: oral, intravenous injection.
In another preferred example, the administration dosage is 50-1000mg/ days, preferably, being 100-500mg/ days, more
Goodly, it is 200-400mg/ days.
Fifth aspect present invention provides a kind of method of the candidate compound of screening inhibition HDAC, comprising steps of
(a) candidate compound is added into the cell culture system of infection β subfamily herpesviral, as experimental group；
It is added without the candidate compound into the cell culture system of infection β subfamily herpesviral, as a control group；
(b) virus titer in measurement experiment group and control group,
Wherein, when the virus titer I1 in experimental group is substantially less than control group I0, then illustrate, the candidate compound is suppression
The candidate compound of HDAC processed.
In another preferred example, described be substantially less than refers to I1≤1/2I0, preferably, I1≤1/10I0
In another preferred example, (b) described in measurement virus titer include in measurement experiment group and cellular control unit
GFP amount of fluorescence and fluorescence are strong and weak；
Wherein, when in experimental group amount of fluorescence and intensity I1 be substantially less than control group I0, then illustrate, the candidate compound
Object is the candidate compound for inhibiting HDAC.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the chemical structural formula of Vorinostat.
Fig. 2 shows various concentration Vorinostat to the inhibition efficiency chart (MOI 0.1) of HCMV virus.
Fig. 3 shows the viral DNA quantitative fluorescent PCR measurement (MOI 0.3) of HCMV after 5 μM of Vorinostat processing.
Fig. 4 shows the viral growth curves (MOI3) of high infection multiplicity HCMV after 5 μM of Vorinostat processing.
Fig. 5 shows the viral growth curves (MOI0.03) of low infection multiplicity HCMV after 5 μM of Vorinostat processing.
After Fig. 6 shows 5 μM of Vorinostat processing, (MOI 3) is detected to the Western Blot of HCMV virus protein.
After Fig. 7 shows 5 μM of Vorinostat processing, (MOI 0.5) is observed to the fluorescence localization at HCMV virus assembly center.
Fig. 8 shows toxicity of the Vorinostat to HF cell of various concentration.
Fig. 9 shows that 10 μM of NSC-3852, CI-994 and BML-281 can significantly inhibit HCMV duplication.
Specific embodiment
The present inventor by extensive and in-depth research, for the first time it was unexpectedly observed that histon deacetylase (HDAC) inhibitor (such as
Vorinostat) duplication and assembling that β subfamily herpesviral (especially human cytomegalovirus) can be inhibited significantly, thus effectively
Destroy the growth of virus.In addition, it is demonstrated experimentally that the hdac inhibitor of low concentration can have height to β subfamily herpesvirus infection
The inhibiting rate of effect inhibits efficiency up to 99.69-99.97%.Therefore, using hdac inhibitor as clinical treatment β subfamily
The active drug of herpesvirus infection.On this basis, the present invention is completed.
Histon deacetylase (HDAC) inhibitor (HDACIs)
Histon deacetylase (HDAC) inhibitor (HDACIs) is a kind of compound, there is interference and histon deacetylase (HDAC)
Function.Histon deacetylase (HDAC) inhibitor is commonly divided into two major classes: NAD+-Dependent enzyme and Zn2+Dependent enzyme.Zn2+According to
Relying property protease includes HDACsI, II, IV subtribe；NAD+-Dependent enzyme is mainly HDACs III subtribe.DNA methylase inhibitor
Enzyme inhibitor improves the approach such as the isogenic expression of p21 by the degree of acetylation of the intracellular histone of increase, inhibits swollen
The proliferation of oncocyte, Cell differentiation inducing activity and (or) apoptosis.Histon deacetylase (HDAC) inhibitor has become neoplasm targeted therapy
The new hot spot of research, to tumor cell migration, invasion, the inhibiting effect of transfer and Antineoplastic angiogenesis effect also demonstrate,proved
It is real.
As used herein, term " the compounds of this invention ", " inhibitor of the present invention ", " hdac inhibitor ", " present invention activity
Ingredient " is used interchangeably, and refers both to histon deacetylase (HDAC) inhibitor, it is preferable that the histon deacetylase (HDAC) inhibits
Agent is Vorinostat.
Histon deacetylase (HDAC) inhibitor for use in the present invention is not particularly limited, and can be any commercially available group egg
White deacetylase inhibitor.
A kind of preferred histon deacetylase (HDAC) inhibitor is Vorinostat.The entitled N- hydroxy-n of Vorinostat chemistry '-
Phenyl suberamide, english abbreviation SAHA, white powdery solids, molecular formula C14H20N2O3, molal weight 264.32g/
mol.Vorinostat can inhibit HDAC 1, HDAC 2 and HDAC 3 and 6 enzymatic activity of HDAC in nanomolar concentrations.At certain
In a little cancer cells, Vorinostat, which reduces HDAC activity, to be helped to slow down or stop the gene activation of growth of cancer cells.Vorinostat
The HIV of energy activating dormant infection, abolishes the latency in latent virus library, then cooperate effective antiviral therapy, to cure HIV
There is provided may.
It is demonstrated experimentally that Vorinostat has the effects that reduce HCMV viral dna replication, obvious inhibition virus infection.
Further, it is also possible to be used for histon deacetylase (HDAC) inhibitor of the invention further include:
8- hydroxyl -5- nitroquinoline (NSC-3852)；
4- acetylaminohydroxyphenylarsonic acid N- (2'- aminophenyl)-benzamide (Tacedinaline, CI-994)；
N- hydroxyl -7- [5- (4- three-level butoxy glycosyl aminobenzene) -3- isoxazole formamide] heptamide (N-Hydroxy-
7- [5- (4-tertbutoxycarbonylaminophenyl) -3-isoxazolecarboxamido] hep tanamide,
BML-281)。
It is demonstrated experimentally that a variety of histon deacetylase (HDAC) inhibitors can duplication to HCMV it is inhibited.
Herpesviral and β subfamily herpesviral
Herpesviral can be divided into tri- subfamilies of α, β, γ, be distrand DNA virus.α subfamily herpesviral (such as herpe simplex disease
Poison, one herpes zoster virus of varicella) growth rate is fast, cause cytopathy.β subfamily herpesviral (such as cytomegalovirus), it is raw
Long period is long, and infection cell forms giant cell.γ subfamily herpesviral (such as Epstein-Barr virus), the target cell of infection are that lymph sample is thin
Born of the same parents can cause lymphocytic hyperplasia.The host range of β subfamily herpesvirus infection is extensive, can infect the mankind and other vertebrates.Draw
That plays mankind's generation has 7 kinds of herpesvirals: herpes simplex virus type 1, herpes simplex virus type 2, varicellazoster virus, EB
Virus, cytomegalovirus, human herpes virus-6, Human Herpesvirus 7.
Symptom caused by herpesviral has self limiting more, in general two weeks can self-healing, the purpose for the treatment of is mostly to prevent from recurring.
Primary treatment regimen includes: systemic therapy, local treatment and Chinese traditional treatment.The principle of systemic therapy includes inactivation of viruses and adjusting
It is immune, it with acyclovir intravenous drip or can take orally, beautiful pearl prestige is oral, interferon intramuscular injection, II muscle of interleukins note
It penetrates.
Wherein, cytomegalovirus infection is to thymus development and splenocyte, mononuclear phagocytic cells, NK cell and CTL cell
Function has significant impact, the immune function of body can be caused to reduce, especially cellular immune function decreased.Human cytomegalovirus disease
Poison is most common, the maximum a kind of virus of harm in intrauterine infection, and there is no effective treatment method so far.
Pharmaceutical composition and method of administration
The present invention also provides a kind of for treating the pharmaceutical composition of β subfamily herpesviral, the pharmaceutical composition
In histon deacetylase (HDAC) inhibitor and pharmaceutically acceptable carrier containing safe and effective amount.In addition, drug of the present invention
Drug, such as Ganciclovir, acyclovir, interferon, interleukin etc. of the composition also containing other treatment herpesviral.
In general, these substances can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium,
Middle pH is usually about 5~8, and preferably pH is about 6~8, although pH value can be with the property and disease to be treated for being formulated substance
Disease and be varied.Prepared pharmaceutical composition can be administered by conventional route, including (but being not limited to):
Oral, intramuscular, intravenous, subcutaneous, intradermal or local administration.
When histon deacetylase (HDAC) inhibitor of the present invention is used for such use, can pharmaceutically be connect with one or more
Carrier or the excipient mixing received, such as solvent, diluent, (can preferably waited with sterile injectable solution or suspension form
Seep and contain about 0.05-5% suspending agent in medium) carry out parenteral routes.It is mixed for example, these pharmaceutical preparations can contain with carrier
About 0.01-99%, the active constituent of more preferably about 0.1%-90% (weight).
The effective dose of active constituent used can be with the tight of compound used, the mode of administration and disease to be treated
Weight degree and change.However, usually when the compound of the present invention is given daily with the dosage of about 0.5-1000mg/kg the weight of animals
When, satisfactory effect can be obtained, is preferably given daily with 1-4 dosage.For most of large mammal,
Daily accumulated dose is about 1-100mg/kg, is preferably about 2-100mg/kg.This dosage is adjusted most preferably to control to provide
Treat response.For example, dosage can be increased or decreased pari passu daily by an urgent demand for the treatment of situation.
The compounds of this invention or pharmaceutical composition preferably pass through the approach administration such as intravenous.Liquid carrier include: sterile water,
Polyethylene glycol, propylene glycol, ethyl alcohol, dimethyl sulfoxide, nonionic surface active agent etc., if be suitble to active constituent characteristic and
Required specific administration mode.Usually used adjuvant can also advantageously be included in preparation pharmaceutical composition, such as anti-corrosion
Agent and antioxidant such as vitamin E, vitamin C, citric acid, BHT and BHA.
The medicament forms for being adapted to injection include: aseptic aqueous solution or dispersion liquid and aseptic powder (for extemporaneous preparation of sterile
Inject solution or dispersion liquid).In all situations, these forms must be sterile and must be fluids to be easy to syringe row
Fluid out.It must be stable under conditions of manufacture and storage, and must be able to prevent the pollution of microorganism (such as bacterium and fungi)
It influences.Carrier can be solvent or decentralized medium, wherein containing as water, alcohol (such as glycerol, propylene glycol and liquid polyethylene glycol), it
Properly mix object and vegetable oil.
Inhibit the method for β subfamily herpesviral
The present invention also provides a kind of methods for inhibiting β subfamily herpesviral in vivo or in vitro.In general, can be in histone
In the presence of deacetylase inhibitor, cultivates β subfamily herpesviral or infect the cell of β subfamily herpesviral, and observe virus
Duplication, assembling situation.
β has been infected further, it is also possible to which pharmaceutical composition or histon deacetylase (HDAC) inhibitor of the invention are applied to
The object of subfamily herpesviral, to inhibit the infection of β subfamily herpesviral.Such as it is applied by approach such as oral, intravenous injections
For object in need for the treatment of.
In vitro in test, the concentration of available histon deacetylase (HDAC) inhibitor is 1-100 μM, preferably, being 1-
50 μM, be 2-10 μM more preferably.Those skilled in the art can test according to the conventional method of external concentration and the prior art
And the usable range of decision in animal body.
Sieve prescription method
The present invention also provides a kind of methods for screening hdac inhibitor, comprising steps of
(a) candidate compound is added into the cell culture system of infection β subfamily herpesviral, as experimental group；
It is added without the candidate compound into the cell culture system of infection β subfamily herpesviral, as a control group；
(b) virus titer in measurement experiment group and control group,
Wherein, when the virus titer I1 in experimental group is substantially less than control group I0, then illustrate, the candidate compound is suppression
The candidate compound of HDAC processed.
In another preferred example, (b) described in measurement virus titer include in measurement experiment group and cellular control unit
GFP amount of fluorescence and fluorescence are strong and weak；
Wherein, when in experimental group amount of fluorescence and intensity I1 be substantially less than control group I0, then illustrate, the candidate compound
Object is the candidate compound for inhibiting HDAC.
Therefore, it is sieve medicine target spot that the present invention, which sieves prescription method with the mechanism that HDAC inhibits β subfamily herpesviral, can screen,
Develop new hdac inhibitor.
Beneficial effects of the present invention
The histon deacetylase (HDAC) inhibitor for being originally intended to oncotherapy is used for controlling for β subfamily herpesviral by the present invention
Treat, especially Vorinostat can by reduce human cytomegalovirus β subfamily herpesviral duplication and assembling to inhibit its proliferation and
Growth, the clinical treatment for β subfamily herpesviral provide new approach.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
The predissolve of 1 Vorinostat of embodiment (SAHA) stores
The mother liquor of Vorinostat stores: precise Vorinostat is simultaneously dissolved in DMSO, concentration 10mM,
1.5mL centrifuge tube is taken, every pipe dispenses 20 μ L, stores in -20 DEG C.
2 titre of embodiment tests the relationship of the concentration and its anti-virus ability that determine Vorinostat
The preparation of culture medium: 500mL DMEM, 50mL FBS, 500 μ L antibiotic Ps/S mix.
The above culture medium cultivates HF cell, and 10cm culture dish culture cell, every ware uses culture medium 10mL every time.One
A 10cm culture dish culture HF cell, is covered with to it, it is digested with 1mL pancreatin.To assign in 12 orifice plates, then it is added to
It is mixed in 15mL culture medium, is assigned in each hole by every hole 1mL, be placed in 5%CO2, it cultivates 40 hours in 37 DEG C of constant incubators, it can
For virus infection；It to assign in 96 orifice plates, is then added in 30mL culture medium and mixes, assign in each hole, set by every 100 μ L of hole
In 5%CO2, cultivate 40 hours in 37 DEG C of constant incubators, can be used for virus titer test.
The dilution of Vorinostat uses: the cell concentration cultivated as needed calculates the volume of required culture medium, according to culture
The volume of base is added certain volume Vorinostat mother liquor, it is made to be diluted to corresponding concentration.
Virus freezes: the cell of HCMV virus infection is cultivated 20 days or so, will appear the virus cracked out in culture medium
Particle collects culture medium, and 4000rpm is centrifuged 20min, draws supernatant, -80 DEG C freeze.
It freezes the recovery and infection of virus: virus is taken out from -80 DEG C, 37 DEG C of 1~2min of water-bath melt viral sample, surpass
1min is swung in acoustic shock is resuspended virion sufficiently, and the culture volume that infection multiplicity and culture cell as needed needs calculates
The viral sample volume needed out is taken out and is mixed with culture medium.
Virus titer experimental procedure:
The cell WT-GFP virus infection of (1) 12 orifice plate, while Vorinostat or DMSO processing, every kind of condition being added to set two
A parallel hole.By added with the culture medium of virus and corresponding concentration Vorinostat, every hole adds 300 μ L, infects 2h.
(2) culture medium is removed after having infected, every hole is changed to 1mL and contains only corresponding concentration Vorinostat or DMSO without virus
Culture medium, be statically placed in 5%CO2, 37 DEG C of constant incubator cultures.
(3) culture will receive corresponding mark added with the culture medium of corresponding concentration Vorinostat to required number of days after infecting
In 1.5mL EP pipe, 4000rpm, 5min centrifugation discard lower layer's cell fragment, supernatant are set in -80 DEG C of refrigerators and is saved.
(4) to 96 orifice plate cell culture 40h, when can carry out virus infection, the viral sample frozen is melted in 37 DEG C,
Ultrasonic vibration 30S.
(5) 10 times of gradient dilutions are carried out to the culture medium containing virus.This experiment tests 7 dilutions to viral supernatants, i.e.,
10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7.Dilution is as follows, to a pipe sample, first prepares 7 EP pipes, respectively plus 1mL
DMEM marks upper " -1, -2, -3, -4, -5, -6, -7 " by dilution on mark respectively, and 110 μ L of supernatant is taken to be added to " -1 " mark
Guan Zhong is vortexed and mixes, and 110 μ L of mixing liquid is taken to be added in the pipe of " -2 " mark, is vortexed and mixes, and successively dilutes the sample of corresponding concentration
Product.
The sample of one dilution is added to by every 50 μ L of hole 12 of a row by one row of (6) 96 orifice plate totally 12 holes
The sample of 7 dilutions is so added in 96 orifice plates, dilution is corresponded in mark by Kong Zhong, in 37 DEG C, 5%CO2Constant temperature training
It supports in case stationary culture 2 weeks.
(7) after two weeks, 96 orifice plates is taken under the microscope, to record the hole that each dilution has virus fluorescence in fluorescence microscopy
Quantity obtains the virus titer of Supernatant samples by conversion.
The concentration of Vorinostat and the relationship of its anti-virus ability are studied, 0 μM of 5 concentration gradients are set, 0.5 μM, 1 μM, 2
μM, 5 μM, virus infection plural number (MOI) 0.1 is chosen, collects the culture medium of 6 days (6dpi) after infection, carries out virus titer experiment
And suppression curve is drawn according to Titer Data.
Experimental result shows, also increases accordingly with the ability of the raising of concentration of Vorinostat its anti-HCMV, and it is 5
To the inhibiting rate of virus infection more than 99.69% when μM concentration.
Vorinostat is about 0.45 μM to the ic50 of HCMV, ic90 is about 1.5 μM.
Influence of embodiment 3 fluorescence quantitative PCR detection, the 5 μM of Vorinostats to HCMV viral dna replication.
The preparation of 2 × digestion buffer (digestion buffer): NaCl 1.1688g, Tris 0.24228g, EDTA
1.8612g, SDS 1g add sterile water to 100mL, mix, be stored at room temperature.
The preparation of 7.5M ammonium acetate: clean 250ml beaker is first added sterile water about 30ml, weighs ammonium acetate 57.81g,
Magnetic stirring apparatus mixes, and is settled to 100ml, is filtered with 0.22 μm of filter into two 50ml centrifuge tubes, be stored at room temperature.
The extraction steps of cell DNA and viral DNA:
(1) point 12 orifice plate cells as described in example 2, after culture 40h, with WT-GFP virus infection, while Jia Fulinuo
He is handled, and timing waits infecting receives sample after a certain period of time.
(2) to after the time, corresponding aperture sample is collected, every hole is washed three times with 1 × PBS that 1ml is pre-chilled.
(3) 100 μ L 2 × digestion buffer are added in every hole, and cell is scraped off culture plate with cell scraper, are added
1.5mL centrifuge tube.
(4) every hole is washed one time with 100 μ 2 × digestion of L buffer, and 1.5mL centrifuge tube is added (can be by cell cryopreservation
In -80 DEG C).
(5) 200 μ L TE of every solencyte addition (contain 100 μ g/ml Proteinase Ks, Proteinase K is with 20mg/ml, that is, 200x storage
It deposits).
(6) sample is incubated overnight in 55 DEG C.
(7) isometric phenol: chloroform: isoamyl alcohol (400 μ L) is added in every pipe, and rotation mixes 10 minutes.
(8) it is centrifuged 1700g (micro centrifuge 4,400rpm), 10min.Supernatant is transferred in new pipe.
(9) RNase A to 100 μ g/mL (100 ×), 37 DEG C of incubation 1h is added in every pipe.
(10) phenol: chloroform: isoamyl alcohol extraction repeats step 7,8.
(11) isometric chloroform is added in every pipe.
(12) it is centrifuged 1700g (micro centrifuge 4,400rpm), 10min.Supernatant is transferred in new pipe.
(13) the 7.5M ammonium acetate of 1/2 volume, 1 μ L glycogen is first added in every pipe, pats and mixes, adds 2 times of volumes
100% ethyl alcohol precipitates DNA.
(14) with maximum (top) speed in micro centrifuge, 4 DEG C are centrifuged 30 minutes, abandon supernatant.
(15) with 500 μ L, 70% ethanol washing, 4 DEG C are centrifuged 5 minutes.After precipitating is dry, dissolved with sterile water, 4 degrees Celsius
Storage is overnight.
The preparation of quantitative fluorescent PCR reaction system (20 μ L): SYBR Premix Ex Taq II (2X) 10 μ L, primer are mixed
Even liquid (positive+reversed) (10 μM) 1.6 μ L, ROX Reference Dye (50X) 0.4 μ L, the 8 μ L of DNA extracted.
Study influence of 5 μM of Vorinostats to HCMV viral dna replication.MOI 0.3 is chosen, as described in above-mentioned steps 1
Cell is managed, collects 4hpi, for 24 hours pi, the sample of 48hpi, 72hpi extract DNA, and the qPCR for designing the vertical early gene IE of virus draws
Object, quantitative fluorescent PCR determine the amount of viral DNA, design the qPCR primer of cell actin gene, and quantitative fluorescent PCR determines cell
Quantity, as internal reference.By comparing the amount of viral DNA under different condition and the ratio size of cell quantity, different condition is obtained
Influence of the lower Vorinostat to viral dna replication.
Experimental result shows that 5 μM of SAHA inhibit about 20 times to viral dna replication.
Inhibitory effect of 4 titre of embodiment experiment, the 5 μM of Vorinostats of detection to high infection multiplicity HCMV.
5 μM of Vorinostats are studied to the inhibitory effect of high infection multiplicity virus.MOI 3 is chosen, is infected as described in Example 2
And 12 orifice plate cells are handled with Vorinostat, 2,4,6 days metainfective culture mediums are collected respectively, and virus is carried out as described in example 2
Titre tests and makes growth curve according to Titer Data.
Experimental result shows that 5 μM of Vorinostats have 99.957%~99.975% in the HCMV infection of high infection multiplicity
Inhibitory effect.
Inhibitory effect of 5 titre of embodiment experiment, the 5 μM of Vorinostats of detection to low infection multiplicity HCMV.
5 μM of Vorinostats are studied to the inhibitory effect of high infection multiplicity virus.MOI 0.03 is chosen, as described in Example 2
Infect simultaneously with Vorinostat handle 12 orifice plate cells, collect 5,10,15 days metainfective culture mediums respectively, as described in example 2 into
Row virus titer tests and makes growth curve according to Titer Data.
Experimental result shows that 5 μM of Vorinostats have 99.973% inhibition effect in the HCMV infection of low infection multiplicity
Fruit.
6 Western Blot of embodiment detects influence of 5 μM of Vorinostats to viral lifecycle.
Study influence of 5 μM of Vorinostats to viral lifecycle.IE1, IE2 are HCMV early expression albumen immediately,
UL38 and UL117.5 is early protein, and pp28 and pp71 are late protein, and wherein pp28 is centrally formed related with virus assembly,
It is simultaneously also the labelled protein at virus assembly center.MOI 3 is chosen in experiment, and collects infection in 8,24,48 and 72 hours and dosing
The sample detection of object processing.
The processing of Western Blot laboratory sample and detecting step:
(1) 6 orifice plates culture HF cell infects and adds Vorinostat processing.
(2) to after receiving the sample time, culture medium is removed, PBS is added to wash twice.
(3) plus 100 μ L cell pyrolysis liquids are to every hole, scrape off cell and cell lysate with cell scraper, are drawn onto 1.5mL
In centrifuge tube, 100 DEG C are boiled 10min, excusing from death concussion 30s.
(4) SDS-PAGE electrophoresis, 60V walk glue 150min.
(5) wet process transferring film, 30V constant pressure transferring film are stayed overnight.
(6) 5% milk (1 × TBST dilution) room temperature closes 1h.
(7) primary antibody incubation at room temperature 2h (5% milk dilution, dilution is depending on different antibodies).
(8) 1 × TBST wash film 5 times, each 5min.
(9) secondary antibody (1 × TBST dilution) is incubated at room temperature 1h.
(10) 1 × TBST wash film 5 times, each 5min.
(11) ECL fluorescent marker is incubated for 5min, darkroom exposure.
Experimental result shows that the 72h to after infecting, 5 μM of Vorinostats have inhibition to the protein content of virus, wherein right
The effect of UL117.5 and pp28 is particularly evident.
7 immunofluorescence of embodiment observes influence of 5 μM of Vorinostats to virus assembly center.
Study influence of 5 μM of Vorinostats to virus assembly center.Such as embodiment 6, pp28 is the mark at virus assembly center
Remember albumen (Fig. 7 is red).Normal HCMV assembling center is spherical in shape by nucleus, and therefore nucleus is also deformed into kidney shape.
DAPI contaminates nucleus (Fig. 7 blue), and GFP marks the cell (Fig. 7 green) of virus infection.MOI 0.5 was chosen in experiment, to 96 hours
It infects and the immunofluorescent staining of drug-treated is added to observe.
Immunofluorescence experiment step:
(1) cell is cultivated on 24 orifice plate coverslips.Before cell dissociation, with tweezers folder coverslip to 24 orifice plates, by 1
10cm plate is digested into 18-20mlDMEM culture medium, every 500 μ L bed board of hole, should not acutely be rocked in order to avoid the poly- heap of cell.
(2) culture medium is sucked, is washed 2 times with PBS.
(3) cell 20min is fixed with 500 μ L, 2% paraformaldehyde.
(4) it is washed 2 times with PBS, is placed 1 minute every time.Coverslip after fixation can be put one week at 4 degree.
(5) with the 500 permeabilized cell 15min of μ L 0.1%Triton X-100.
(6) it is washed 2 times with PBS.
(7) with 500 μ L block buffer (in 5%FBS in PBS, 9.5ml PBS plus 500 μ L FBS) closing
20min。
(8) on 40 μ L pp28 (1:200) primary antibodies (being diluted with block buffer) plus parafilm film, coverslip has
The one side of cell is incubated for 1 hour with primary antibody.
(9) it is washed 3 times with PBS.
(10) 1 time 5min is washed with 500 μ L block buffer.
(11) it (is diluted with block buffer) with 50 μ L secondary antibodies, is protected from light incubation 1 hour, operates with step 8.
(12) it is washed 3 times with PBS.
(13) it with the DAPI of 300 μ L 1:5000 dilution (PBS), contaminates cell 10 minutes.
(14) it is washed 2 times with PBS.
(15) it is infiltrated once in distilled water H2O, blots excessive moisture on coverslip with lens wiping paper.
(16) it by coverslip lid to the glass slide for having 8 μ L of Prolong Gold, marks, is protected from light and stood
Night.
(17) coverslip is sealed with nail polish, it is 30 minutes dry, film-making is kept in dark place.
(18) the micro- sem observation film-making of fluorescent co-location.
Experimental result shows that the cell virus assembling center of normal infection HCMV normally forms, and core is kidney-shaped；5 μM of volts are vertical
After his processing of promise, virus assembly center cannot be formed, and nucleus still at spherical or elliposoidal, inhibits the shape at virus assembly center
At.As it can be seen that 5 μM of Vorinostats can inhibit the formation at virus assembly center.
Embodiment 8 detects toxicity of the Vorinostat to HF cell of various concentration.
After Vorinostat processing 3 days of various concentration are added in HF cell, propidium iodide (Propidium is utilized
Iodide, PI) and Hoechst dyeing, Hoechst can dye with penetrating cell film and indicate all cell number, PI can be penetrated
The cell membrane of lost integrity, the cell that dyeing instruction will be dead.The ratio characterization of compound of PI/Hoechst is to cell
Toxic effect.PBS handles positive control of 4 hours cells as experiment.Experimental result shows that 5 μM of Vorinostat is to thin
The toxic effect of born of the same parents is 1.9%, and 10 μM of Vorinostat is 2.4% to the toxic effect of cell.
Inhibitory effect of the different hdac inhibitors of embodiment 9 to HCMV.
Different hdac inhibitors is studied to the inhibitory effect of low infection multiplicity virus.
Choose MOI 0.3, as described in Example 2 infection and with 10 μM of different hdac inhibitors (including NSC-3852,
CI-994, BML-281) 12 orifice plate cells of (being purchased from Enzo) processing, it takes pictures under fluorescence microscope within the 8th day after infection.It is big and small
Green fluorescent protein, the speed of number and intensity the reaction virus replication of green fluorescent protein are embedded in cellular virus genome.
Experimental result shows (Fig. 9) that 10 μM of NSC-3852, CI-994 and BML-281 can significantly inhibit HCMV duplication.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (8)
1. a kind of method of the inhibition β subfamily herpesviral of external non-therapeutic, which is characterized in that in the presence of hdac inhibitor
Under, it cultivates β subfamily herpesviral or infects the cell of β subfamily herpesviral, wherein the cell includes human fibroblasts,
Human embryonic lung diploid fibroblast, Human RPE Cells in Vitro；
Wherein, the hdac inhibitor includes Vorinostat (Vorinostat, SAHA), 8- hydroxyl -5- nitre
Base quinoline (NSC-3852), 4- acetylaminohydroxyphenylarsonic acid N- (2'- aminophenyl)-benzamide (CI-994), N- hydroxyl -7- [5- (4-
Three-level butoxy glycosyl aminobenzene) -3- isoxazole formamide] heptamide (BML-281)；
And the β subfamily herpesviral is human cytomegalovirus (CMV).
2. the method as described in claim 1, which is characterized in that the hdac inhibitor is N- hydroxy-n '-phenyl suberoyl
Amine.
3. the method as described in claim 1, which is characterized in that the effective quantity of the hdac inhibitor is 1-100 μM.
4. the method as described in claim 1, which is characterized in that the inhibition β subfamily herpesviral includes inhibiting β subfamily blister
The duplication and/or assembling of exanthema virus.
5. the method as described in claim 1, which is characterized in that hdac inhibitor there are while, there is also anti-β subfamily blisters
Exanthema cytotoxic drug, wherein the anti-β subfamily herpesvirus medicament is selected from the group: Ganciclovir, acyclovir, interferon and
Interleukin.
6. a kind of method that screening inhibits the candidate compound of HDAC, which is characterized in that comprising steps of
(a) candidate compound is added into the cell culture system of infection β subfamily herpesviral, as experimental group；To sense
It contaminates in the cell culture system of β subfamily herpesviral and is added without the candidate compound, as a control group；
(b) virus titer in measurement experiment group and control group；
Wherein, when the virus titer I1 in experimental group is substantially less than control group I0, then illustrate, the candidate compound is to inhibit
The candidate compound of HDAC；
Wherein the cell includes human fibroblasts, human embryonic lung diploid fibroblast, and human retinal pigment's epithelium is thin
Born of the same parents；
Wherein, the β subfamily herpesviral is human cytomegalovirus (CMV).
7. method as claimed in claim 6, which is characterized in that described being substantially less than refers to I1≤1/2I0.
8. method as claimed in claim 6, which is characterized in that the measurement virus titer described in (b) includes measurement experiment group
With the GFP amount of fluorescence and fluorescence power in cellular control unit；
Wherein, when in experimental group amount of fluorescence and intensity I1 be substantially less than control group I0, then illustrate, the candidate compound is
Inhibit the candidate compound of HDAC.
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| SG11202113215UA (en) | 2019-05-31 | 2021-12-30 | Viracta Subsidiary Inc | Methods of treating virally associated cancers with histone deacetylase inhibitors |
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