CN104548060A - Method for adjusting TAK1 (transforming growth factor-activated kinase 1) activity by compound and application of compound to treatment of hand-foot-mouth disease - Google Patents

Abstract

The invention provides a method for identifying whether a compound is used for adjusting the TAK1 (transforming growth factor-activated kinase 1) activity of a mammal, and an application of the compound-2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) and/or a physiologically acceptable salt and a pharmaceutically acceptable carrier of the compound to preparation of drug composition. The drug composition is used for treating a hand-foot-mouth disease caused by enteroviruses 71 or Coxsackie viruses A16 (CVA16). The experimental research shows that m-3M3FBS is used for treatment, so that TAK1 phosphorylation and the activity of MAP kinase and NF-kB (nuclear factor-kB) pathways in dsRNA (double-stranded ribonucleic acid)-stimulated macrophages can be remarkably inhibited, expression of proinflammatory cytokines including TNF (tumor necrosis factor), IL-6 (interleukin-6) and IL-12 (interleukin-12) in poly(I:C)-stimulated macrophages can be remarkably reduced, clinical scores of CVA16-infected mice are reduced, the survival time of the CVA16-infected mice is prolonged, and histopathologic development of the CVA16-infected mice in lung and muscle tissues is reduced.

Description

Compound regulates the method for TAK1 activity and this compound to be used for the treatment of the application of hand foot mouth disease

Technical field

The present invention relates to a kind of compound regulates the method for TAK1 activity and this compound for the preparation of pharmaceutical composition and the application thereof for the treatment of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16.

Background technology

Hand foot mouth disease (Hand, foot and mouth disease, HFMD) be [with COxsackie A group 16 type (CVA16) by enterovirus, enterovirns type 71 (EV71) is common] acute infectious disease that causes, be usually expressed as with herpangina, throat pain and heating for feature gentleness with self-limited disease (Huang, C.C., C.C.Liu, Y.C.Chang, C.Y.Chen, S.T.Wang, and T.F.Yeh.1999.Neurologiccomplications in children with enterovirus 71infection, N Engl J Med.341:936-942).Aseptic meningitis, BBE and acute hemorrhagic necrotizing pancrease (a kind of poliomyelitis is like syndromes) can be there is in minority HFMD case.

The HFMD caused by the enterovirus of such as CVA16 with EV71 brings into millions of the infecteds and hundreds of death, and be considered to now important communicate illness (Solomon, T., P.Lewthwaite, D.Perera, M.J.Cardosa, P.McMinn, and M.H.Ooi.2010.Virlogy, epidemiology, pathogenesis, and control of enterovirus 71.The LancetInfectious Diseases.10:778-790).Do not have effective vaccine or specific antiviral therapy at present, therefore developing is effectively very urgent for the prevention of hand foot mouth disease or treatment means.

It is that the morbidity of HFMD is former that high-caliber IL-6 is in the news.Although the pro-inflammatory cytokine of more brought out after pathogenic agent stimulates should activate the reaction of acute phase and the stimulation (Kanda of the T cell removed for pathogenic agent and B cell, T., J.E.McManus, R.Nagai, S.Imai, T.Suzuki, D.Yang, B.M.McManus, and I.Kobayashi.1996.Modification of viral myocarditis in mice byinterleukin-6.Circ.Res.78:848-856; Kopf, M., H.Baumann, G.Freer, M.Freudenberg, M.Lamers, T.Kishimoto, R.Zinkernagel, H.Bluethmann, and G. 1994.Impaired immune and acute-phase responses in interleukin-6-deficient mice.Nature.368:339-342), the evidence collected but shows, the pro-inflammatory cytokine of excessive level can overwhelm Immune defense, and causes serious immune-inflammatory disease.

Some clinical studyes show, bringing out of a large amount of pro-inflammatory cytokine is that EV71 virus infection causes serious pathogenic factor.In fact, infect in the serum and celiolymph (cerebralspinal fluid, CSF) gathered in patient from EV71 and contain high-caliber pro-inflammatory cytokine, as IL-6, continue to be in the news, such as:

1.Lin,T.Y.,L.Y.Chang,Y.C.Huang,K.H.Hsu,C.H.Chiu,and K.D.Yang.2002.Different proinflammatory reactions in fatal and non-fatal enterovirus 71infections:implications for early recognition and therapy.Acta Paediatr.91:632-635；

2.Lin,T.Y.,S.H.Hsia,Y.C.Huang,C.T.Wu,and L.Y.Chang.2003.Proinflammatory cytokine reactions in enterovirus 71infections of the centralnervous system.Clin Infect Dis.36:269-274；

3.Wang,S.M.,H.Y.Lei,M.C.Huang,L.Y.Su,H.C.Lin,C.K.Yu,J.L.Wang,and C.C.Liu.2006.Modulation of cytokine production by intravenousimmunoglobulin in patients with enterovirus 71-associated brainstemencephalitis.J.Clin.Virol.37:47-52；

4.Wang,S.M.,H.Y.Lei,L.Y.Su,J.M.Wu,C.K.Yu,J.R.Wang,and C.C.Liu.2007.Cerebrospinal fluid and cytokines in enterovirus 71brain stemencephalitis and echovirus meningitis infections of varying severity.Clin.Microbiol.Infect.13:677-682；

5.Weng,K.F.,L.L.Chen,P.N.Huang,and S.R.Shih.2010.Neuralpathogenesis of enterovirus 71infection.Microbes Infect.12:a505-510；

6.Wang,S.M.,H.Y.Lei,and Liu,C.C.2012.Cytokineimmunopathogenesis of enterovirus 71brain stem encephalitis.Clin DevImmunol.2012:876241。

In mouse, it is the pathogenic factor (Khong of EV71 in baby's mouse model that the IL-6 of continual high levels is in the news, W.X., D.G.Foo, S.L.Trasti, E.L.Tan, and S.Alonso.2011.Sustained high levels of interleukin-6contribute to the pathogenesis ofenterovirus 71in a neonate mouse model.J Virol.85:3067-3076).

But the generation of IL-6 is that how to affect the progress of HFMD still unknown.

Summary of the invention

The present invention solves the aforementioned problems in the prior proposition.

The invention provides a kind of method whether authenticating compound regulates Mammals TAK1 activity, the pro-inflammatory cytokine that wherein TAK1 activity mark the pathogenic agent producing the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16 is brought out plays a crucial role.

Present invention also offers a kind of pharmaceutical composition being used for the treatment of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16, described composition comprises albumen, polypeptide or their activating compounds with phospholipase Cβ 2 activity, and/or the salt that its physiology can tolerate and pharmaceutically acceptable carrier.

For achieving the above object, the present invention is by the following technical solutions:

First aspect of the present invention is to provide a kind of method whether authenticating compound regulates Mammals TAK1 activity, comprises the steps:

Step 1, by mammalian cell compound incubation;

Step 2, the viral and virus antigen composition inductor stimulation by the cell of step 1;

Step 3, the TAK1 measured from the cell of step 2 are active;

Wherein, the pro-inflammatory cytokine that described TAK1 activity mark the pathogenic agent producing the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16 is brought out plays a crucial role.

Preferably, the mammalian cell of described step 1 is rodentine cell, and described rodent is more preferably mouse, is more preferably mouse.

Preferably, the mammalian cell of described step 1 is the cell of the mankind.

Wherein, described cell be selected from pancreatic cell, myocyte, liver cell, nephrocyte, brain cell, adipocyte, scavenger cell any one or multiple, be more preferably scavenger cell.

Mammiferous cell is from the cell culture of mouse.

Preferably, the virus of described step 2 and virus antigen composition be selected from poly I:poly C poly (I:C), H5N1, NDV, CAV16 any one or multiple, be more preferably poly I:poly C poly (I:C).

Wherein, the phospholipase Cβ 2 in described compound activating cell.

Second aspect of the present invention is to provide a kind of pharmaceutical composition being used for the treatment of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16, described composition comprises albumen, polypeptide or their activating compounds with phospholipase Cβ 2 activity, and/or the salt that its physiology can tolerate, and pharmaceutically acceptable carrier or vehicle.

Described compound is preferably 2,4,6-trimethylammonium-N-(meta-3-trifluoromethyl-phenyl)-benzsulfamide (2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, m-3M3FBS).

3rd aspect of the present invention is to provide salt that compound m-3M3FBS and/or its physiology can the tolerate purposes for the preparation of pharmaceutical composition, and this pharmaceutical composition is used for the treatment of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16.

The present invention adopts technique scheme, compared with prior art, has following technique effect:

Experimental study shows, significantly can suppress the activity of map kinase in the phosphorylation of TAK1 and scavenger cell and NF-к В path, also significantly reduce the expression that scavenger cell comprises the pro-inflammatory cytokine of TNF, IL-6 and IL-12 with m-3M3FBS process.And, reduce with m-3M3FBS process the clinical score of mouse that CVA16-infects, also extend the survival of the mouse that CVA16-infects, and reduce their histopathology development in lung and muscle tissue.

Accompanying drawing explanation

In Fig. 1: HFMD sufferer, the PLC β 2 with more high expression level combines appearance with lower p-TAK1 and blood serum IL-6;

(A, B) is from immunoblotting (the IB) ， ﹡ p < 0.05 of the PLC β 2 in the peripheral blood lymphocytes (PBMC) of blank group and HFMD sufferer group;

(C, D) is from IL-6 enzyme linked immunosorbent assay (the ELISA) ， ﹡ p < 0.05 in the serum of HFMD sufferer;

(E, F) is from the immunoblotting (IB) of the PLC β 2 in the PBMC of HFMD sufferer and p-TAK1; Based on often with intensity PLC β 2 and p-TAK1 level are divided into basic, normal, high, and the percentage ratio being sorted in the sufferer of each kind is described in the histogram of (F);

(G) immunoblotting (IB) of the PLC β 2 in the Turnover of Mouse Peritoneal Macrophages of CVA16 one period of fixed time of virus is infected;

(H) immunoblotting (IB) that personal poly (I:C) (10mg/ml) stimulates the PLC β 2 in the lysate of the Turnover of Mouse Peritoneal Macrophages of one period of fixed time is carried out;

(I) wild-type (WT) and TLR3 that personal poly (I:C) (10mg/ml) stimulates one period of fixed time is come ^-/-the immunoblotting (IB) of the PLC β 2 in the lysate of Turnover of Mouse Peritoneal Macrophages;

(J) before stimulating one period of fixed time with poly (I:C) (10mg/ml), the immunoblotting (IB) of the PLC β 2 in the Turnover of Mouse Peritoneal Macrophages of 1 hour is anticipated with p38 inhibitor SB203580;

G, H, I, J representative at least three kinds is independently tested.

Fig. 2: PLC β 2 ^-/-(PLC β 2-is not enough) mouse is more easily subject to the infection of CVA16;

(A, B) stimulates WT or the PLC β 2 of one period of fixed time with poly (I:C) (10mg/ml) ^-/-the Q-PCR of Tnf (A) relative in scavenger cell and IL-6 (B) mRNA level in-site analyzes; 14 the largest WT or PLC β 2 ^-/-10 μ l CVA16 (every mouse 4 × 10 are infected in mouse peritoneal ⁵pUF) 0-5 days;

(C, D, E) is from WT or PLC β 2 ^-/-the Q-PCR of relative Tnf (C) in the muscle tissue of mouse, IL-6 (D) and IL-12 (E) mRNA level in-site analyzes (n=3);

(F) WT or the PLC β 2 of 0 day (infection) or 5 days is infected according to the method described above ^-/-the histopathology figure of Muscle Tissue; Original magnification: 100 × (top) or 200 × (bottom);

(G) expression (n=3) of CVA16 from the replicated rna sample that the muscle tissue of the mouse through aforesaid method process obtains;

(H, I) is through WT or the PLC β 2 of designation method process ^-/-the clinical score (H) of mouse or survival rate (I); These data representative at least three kinds independently tests (mean value and s.e.m) ， ﹡ p < 0.05 ， ﹡ ﹡ p < 0.01.

Fig. 3: PLC β 2 suppresses the activity of MAPKs and NF-к В by target TAK1 ubiquitination;

(A, B) at HEK293T cells with the immunoprecipitation (IP) of the product of cell lysis of TAK1 or the PLC β 2 of hemagglutinin (HA) or Flag and immunoblotting (IB);

(C) from immunoprecipitation (IP) and the immunoblotting (IB) of the lysate of the HEK293T cell of expression Flag-PLC β 1-3 and HA-TAK1;

(D) the external GST precipitation test be combined with GST-TAK1 with the PLC β 2 of Histidine (HIS) label of purifying is used;

(E) immunoprecipitation experiment of the GST-TAK1 of the purification be combined with endogenous PLC β 2;

(F) the endogenous interaction relationship of PLC β 2 and TAK1 in the main scavenger cell stimulated through poly (I:C);

(G) immunoblotting assay of the split product of the HEK293T cell of personal phosphoric acid-TAK1 antibody expression PLC β 2 and TAK1 is carried out;

(H, I) uses phosphoric acid-TAK1 antibody (H) or phosphorylation-antibody (I), carrys out WT or the PLC β 2 that personal poly (I:C) (10mg/ml) stimulates one period of fixed time ^-/-the immunoblotting assay of the lysate of scavenger cell;

(J) exist or lacking immunoprecipitation (IP) and immunoblotting (IB) analysis that PLC β expresses the split product of the HEK293T cell of K63-Ub, TAK1 and TAB1 for 2 times;

(K) WT or PLC β 2 is stimulated with poly (I:C) ^-/-one period of fixed time of mouse, and by the anti-TAK1 immunoprecipitation of product of cell lysis; With anti-Ub (K63) antibody analysis immunoprecipitate;

(L) exist or lacking immunoprecipitation (IP) and immunoblotting (IB) analysis that PLC β expresses the lysate of the HEK293T cell of TAK1 and TAB1 for 2 times; These data representative at least three kinds is independently tested.

Fig. 4: m-3M3FBS alleviates the hand foot mouth disease that CVA16 virus infection causes;

(A, B) come personal DMSO or m-3M3FBS pretreatment of mice source peritoneal macrophage, then use phosphorylation-antibody to poly (I:C) stimulate the split product of the scavenger cell of one period of fixed time carry out immunoblotting (IB) analyze;

(C, D, E) 14 days large WT mouse DMSO or m-3M3FBS pre-treatment also infect 0-5 days with CVA16 subsequently; Q-PCR from the relative Tnf (C) in two groups of muscle tissue, IL-6 (D) and IL-12 (E) mRNA level in-site analyzes (n=3);

(F) from the histopathology figure of two groups of muscle tissues obtained; Original magnification: 100 × (top) or 200 × (bottom);

(G, H) is through the clinical score (G) of the mouse of aforesaid method process or survival rate (H); These data representative at least three kinds independently tests (mean value and s.e.m);

(I) chart describes by viral-induced PLC β 2, by target TAK1 ubiquitination, and negative regulation innate immune response.

Embodiment

Carry out detailed and concrete introduction below by specific embodiment to the present invention, to make better to understand the present invention, but following embodiment does not limit the scope of the invention.

Test materials and method:

Cell cultures and reagent:

By HEK293T cell cultures in DMEM substratum (Hyclone), through 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco) and 100U/ml penicillin and Streptomycin sulphate.HeLa cell and peritoneal macrophage are cultivated in RPMI-1640 substratum, through 10% (v/v) FBS and 100U/ml penicillin and Streptomycin sulphate.Vero cell (African green monkey kidney cell) is cultivated in VP-SFM substratum (Gibco), 4mML-glutaminate (Gibco).These cell cultures are with 5%CO ₂in 37 DEG C of insulation cans of balance.The micro-carriers cell culture bioremediation reported before use, is placed on the CVA16 bacterial strain be separated clinically in Vero cell and breeds.This virus stocks is stored in-80 DEG C.The plaque-forming assay method Test Virus titre of use standard.

Use following antibody:

The anti-K63-of rabbit connects specific poly-ubiquitin (5621), rabbit anti-phosphoric acid TAK1 (Thr187) (4536), rabbit anti-phosphoric acid TAK1 (Ser412) (9339), rabbit anti-phosphoric acid p65 (3033), rabbit anti-phosphoric acid Erk1/2 (9101), rabbit anti-phosphoric acid p38 (9215) and the anti-phosphoric acid Jnk (9251) of rabbit, and these antibody are from CellSignaling Technology;

Rabbit anti-PLC β 2 (sc-206), rabbit anti-phosphoric acid TAK1 (Ser192) (sc-130219) and the anti-TAK1 of rabbit (sc-7162), these antibody are from Santa Cruz Biotechnology;

The anti-TAB1 of rabbit (ab27983, from Abcam); Monoclonal mouse-anti Flag M2 affinity gel (A2220), monoclonal mouse-anti HA (H9658), the anti-HA of rabbit (H6908), the anti-GAPDH of rabbit (SAB2701826) and the anti-Flag of rabbit (F7425), these antibody are from Sigma;

And Protein G Sepharose ^tM4Fast Flow (GE Healthcare).

Use following compounds:

M-3M3FBS (T5699) and SB203580 (P38 inhibitor, S8307), from Sigma.

Plasmid and plasmid construction:

TAK1 and the deletion mutant thereof of the PLC β 2 encode cDNAs and the form of brachymemma and cDNAs coding are inserted in Flag-pcDNA3 carrier.PLC β 2 and TAK1cDNAs is inserted in HA-pcDNA3 expression vector.TAB1 and TAB2 that cDNAs encodes is inserted in pcDNA3 carrier, and the PLC β 2 encoded by cDNAs and TAK1 is inserted in pET28a and p-GEX-4T-1 carrier respectively.

Mouse species:

Homozygous PLC β 2 and TLR3 gene knockout mice are raised under Shanghai Laboratory Animal Center is without specific pathogenic agent condition.14 days large or 6 weeks large PLC β 2 knock out mice and wild-type blank mouse is used in experiment of the present invention.All zooscopies are ratified with the group of use through the Institutional Animal treatment of Tongji University.

The separation of scavenger cell:

Peritoneal macrophage obtains from the mouse after Brewer improvement thioglycollate broth (BD, Sparks, MD) injects 4 days.These cells poly (I:C) (10mg/ml), LPS (100ng/ml), PGN (25 μ g/ml), H5N1 (MOI=1) or NDV (MOI=1) stimulate one period of specifying.

RT-PCR analyzes:

First, these cells are hatched 12 hours when serum-free.After DMSO or m-3M3FBS (10mM) preincubation 30 minutes, these cells poly (I:C) (10mg/ml) stimulates one period of specifying.According to the instruction of manufacturers instruction (Invitrogen), extract total serum IgE with 1ml TRIzol reagent.Then, ReverTra is used qPCR RT Kit (Toyobo, FSQ-101), according to manufacturers instruction instruction, by 1 μ g total serum IgE reverse transcription.LightCycler (Roche, LC480) and SYBR RT-PCRkit (Toyobo, QPK-212) is used to quantitative Real time RT-PCR analysis.Expression values is standardized as those expression values obtained with respective gene Gapdh (coding GAPDH).

Luciferase reporter gene is tested:

Mammal 293T clone is used to luciferase reporter gene test.Cell is in DMEM (glucose content; Bibco, Life Technologies, Grand Island, NY, USA) in cultivate with 10%FBS.Before transfection, cell is accessed in 96 porocyte culture plates.After 24 hours, use Lipofectamine ^tM2000 transfection reagents (Invitrogen, Life Technologies, Grand Island, NY, USA) are by reporter plasmid (every hole 10ng altogether) and show the common transfection of plasmid (every hole 100ng).After transfection after about 48 hours, according to the instruction of manufacturers instruction, at Modulus ^tMthe multi-functional microplate reader of single tube (Turner Biosystems, Sunnyvale, CA, USA) upper use Luciferase AssayReagent II (LAR II) (Promega, Madison, WI, USA), the activity of quantitative Renilla and Photinus pyralis LUC.Transfection is triplicate.Paired t-test is used to determine significant difference.

Western blot test and co-immunoprecipitation reaction:

For there is immunoprecipitation, calcium phosphate-DNA is used to infect method transient transfection in=293T cell altogether.After 48 hours, use PBS washed cell, and subsequently at lysis buffer (20mM Tris (pH7.5), 150mM NaCl, 1%Triton X-100, trisodium phosphate, β-glycerophosphate, EDTA, Na ₃vO ₄, leupeptin) and middle lysis, add 1% protease inhibitor cocktail (Sigma, P8340), 1mMNaF and 1mM Na ₃vO ₄ ⁴⁹.Be positioned on ice after 30 minutes, by lysate with the speed of 13523 × g, centrifugation 15 minutes at 4 DEG C, to remove fragment.By the anti-Flag M2 affinity gel of product of cell lysis, at 4 DEG C incubated overnight.For there is interior raw immunoprecipitation, peritoneal macrophage poly (I:C) stimulates one period of specifying.Cell is cleaved subsequently, and by lysate PLC β 2 antibody and ProteinG Sepharose ^tM4Fast Flow, at 4 DEG C incubated overnight.Centrifugation agarose sample, washs three times by cell lysis buffer solution, and boils 8 minutes with SDS sample-loading buffer.

Gst fusion protein and precipitation test:

By pcr amplification TAK1 and PLC β 2 albumen, and difference subclone is in pGEX-4T1 (Amersham) and pET28a (Novagen) carrier.According to manufacturers instruction instruction, His and gst fusion protein are expressed in BL-21 (DE3) bacterium (Invitrogen).Dissolve according to the method described above in RAW264.7 cell.By the suitable fusion rotein of the His-PLC β 2 albumen equivalent of lysate or purification, hatch 4 hours at 4 DEG C, be attached on gsh pearl.By the centrifugation of peptide pearl, washing, boil 8 minutes with SDS sample-loading buffer, and pass through immunoblotting assay subsequently.

Cell dyeing and confocal microscope:

According to manufacturers instruction instruction, use Lipofectamine ^tM2000 (11668-019, Invitrogen) transfection HeLa cell.Transfection is after 48 hours, with 4% formaldehyde fixed cell 30 minutes at 25 DEG C.Cell is colored, and uses the Leica confocal microscope being equipped with analysis software to detect.

Experimentation on animals:

With syringe by 10 μ l CVA16 (every mouse 4 × 10 ⁵pUF) abdominal injection specified-pathogens free 14 days large WT or PLC β 2 knock out mice.Observe clinical symptom and the death toll of 1 mouse every day, observe 14 days.DMSO or m-3M3FBS (5mg/kg) is expelled in the mouse of CVA16 infection, injects 3 times (infected before 1 day, infect after 2 days and infect after 5 days).

Clinical disease is by following scoring:

0, healthy;

1, flanging fur and bow-backed outward appearance;

2, consumption;

3, limbs fatigue;

4, tetraplegia;

5, dying and dead.

Infect after 5 days, from WT or PLC β 2 gene knockout mice, gather lung, muscle, spleen tissue sample, and be fixed in the buffered formalin (Sigma-Aldriich) of 10% neutrality.These tissues are dyeed by by paraffin embedding, incision, use Yihong stain (H & E).Obtain the bright-field microscope photo of × 100 or × 200 enlargement ratios.All zooscopies are ratified with the group of use through the Institutional Animal management of Tongji University.

Statistical study:

Statistical significance between two groups is checked by two tail student t and two-way ANOVA (ANOVA) is determined.As p < 0.05, be considered to there is marked difference.

Ethics is stated:

Work of the present invention accepts the approval from the clinical ethics council of the attached Xinhua Hospital of pharmaceutical college of Shanghai Communications University, meets the guilding principle of national government and institutional policy.Obtain the Written informed consent from the minor/Children participation person participated in the present invention's research signature under the accompanying of relative, guardian or ward.Ethics Committee clearly have approved the program of this part of letter of consent.

Human sample:

Whole blood sample is obtained from being less than 10 years old large children.Go out 6 children according to ministry of Health of China HFMD Case definition (2008) (http://www.moh.gov.cn/mohbgt/s9503/200812/38494.shtml) clinical diagnosis and suffer from HFMD.In addition, the healthy children of HFMD history that do not have of 5 years old coupling is included as blank group.For carrying out western blot test, according to the instruction of manufacturers instruction, process whole blood sample with red blood cell lysis buffer (eBioscience); Then, remaining cell is dissolved in lysis buffer.

Test result analysis:

From the sufferer sample of blood, drawn that 5 do not have the normal healthy controls group of HFMD history and 6 clinical diagnosises to suffer from HFMD, and analyze these samples with western blot test subsequently.Find that the protein level (a kind of enzyme being used to the key of the conversion of effective signal and white cell activation) of Phospholipase C (phospholipaseC, PLC) β 2 is much higher than control group (Figure 1A and Figure 1B) in HFMD patient.Then, the collection of blood sample is extended to 30 HFMD sufferers, and analyze transforming growth factor-beta activation kinases 1 (transforminggrowth factor-β-activated kinase 1, TAK1), nuclear factor-κB (nuclear factor is regulated, NF-к В) and mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) activity of central element in cell, they are labeled with the path played a crucial role to generation pro-inflammatory cytokine.

According to the level of TAK1 phosphorylation, front 12 samples are divided into two groups.Second group of sufferer has higher levels of TAK1 activity but the PLC β 2 (Fig. 1 E) of lower expression than first group.And, in one group of 30 HFMD clinical sample, find that TAK1 activity and PLC β 2 protein level exist inverse correlation (Fig. 1 F).And second group of HFMD sufferer produces significantly lower IL-6 (Fig. 1 C and Fig. 1 D).These results show, and PLC β 2 can suppress TAK1 active, which reduces the generation of IL-6.

Then, determine whether PLC β 2 is brought out by the infection of CVA16 virus.In fact, the expression (Fig. 1 G) that CVA16 can bring out PLC β 2 is infected.Usually dsRNA (Weber can be produced in virus infection; F.; V.Wagner; S.B.Rasmussen; R.Hartmann, and S.R.Paludan.2006.Double-stranded RNA is produced by positive-strand RNA viruses andDNA viruses but not in detectable amounts by negative-strand RNA viruses.JVirol.80:5059-5064).The generation (Fig. 1 H) also improving PLC β 2 is stimulated with poly I:poly C poly (I:C).But, at TLR3 ^-/-in scavenger cell or wild-type (wild-type, WT) scavenger cell, the rise of PLC β 2 is stimulated to be weakened by poly (I:C), with SB203580 process (Fig. 1 I and Fig. 1 J).These results show, and PLC β 2 can be brought out by TLR3-p38 path by dsRNA.

For checking whether PLC β 2 works to the generation of the pro-inflammatory cytokine that CVA16 brings out, stimulate from PLC β 2 with poly (I:C) ^-/-the scavenger cell of (PLC β 2-is not enough) mouse or WT mouse, and the generation of pro-inflammatory cytokine is analyzed with quantitative RT-PCR assay.After stimulating with poly (I:C), PLC β 2 ^-/-scavenger cell demonstrate and produce higher pro-inflammatory cytokine TNF and IL-6 (Fig. 2 A and Fig. 2 B) than WT scavenger cell.For investigation PLC β 2 function in vivo, by two weeks large WT mouse or PLC β 2 ^-/-mouse CVA16 virus infection 5 days, the expression level quantitative RT-PCR assay of pro-inflammatory cytokine is analyzed.PLC β 2 ^-/-the muscle of mouse has TNF (Fig. 2 C), the IL-6 (Fig. 2 D) significantly higher than WT mouse, the mRNA level in-site of IL-12 (Fig. 2 E), describes the viral-induced expression of proinflammatory factor in PLC β 2 negative regulation body.By two weeks large WT mouse or PLC β 2 ^-/-mouse CVA16 virus infection 5 days, the further functional meaning of investigation PLC β 2 in host anti-virus reaction.The PLC β 2 that CVA16 infects ^-/-mouse has the clinical score (Fig. 2 G and Fig. 2 H) of the virus titer higher than WT mouse and Geng Gao in their muscle tissue.The PLC β 2 that CVA16 infects ^-/-mouse is than WT mouse faster dead (Fig. 2 I).From the histological indications display infecting the isolated lung of CVA16 mouse (not providing) or muscle tissue, PLC β 2 ^-/-mouse has higher histopathology development (Fig. 2 F) than WT mouse.PLC β 2 ^-/-mouse is more easily subject to this statement of facts of the infection of CVA16 virus, and PLC β 2 plays the effect of the former negative regulation agent of the morbidity of In vivo infection CVA16.

For investigating the mechanism of PLC β 2 in the function of the viral-induced generation pro-inflammatory cytokine of adjustment further, whether PLC β 2 is interrelated detected with TAK1.Express in instantaneous infection in Human embryonic kidney (human embryonic kidney, the HEK293T) cell of TAK1 and PLC β 2, TAK1 and PLC β 2 is occurred together in immunoprecipitation (Fig. 3 A and Fig. 3 B).But TAK1 does not have immunoprecipitation (Fig. 3 C) together with other isotype PLC β 1 of PLC β or PLC β 3.Glutathione S-transferase (glutathione S-transferase in vivo, GST) using the recombinant protein through purifying in the precipitator method, finding that TAK1 albumen is with the restructuring His-PLC β 2 through purifying or from raw PLC β 2 united (Fig. 3 D and Fig. 3 E) in RAW264.7 cell.Finally, stimulate the interaction (Fig. 3 F) that improve raw TAK1 and interior raw PLC β 2 in Turnover of Mouse Peritoneal Macrophages with poly (I:C), illustrate that the interaction between TAK1 and PLC β 2 stimulates to rely on.PLC β 2 comprises four territories: PH_PLC, Plcc, C2_2 and PLC-β C.For determining the region be connected with TAK1 in PLC β 2, build the deletion mutant of PLC β 2, and analyze the interaction of they and TAK1.Plcc and C2_2 and TAK1 interacts, and PH_PLC and PLC-β C does not have (for providing).With being consistent, confocal microscope analysis shows result, in HeLa cell overexpression TAK1 and in PLC β 2 mutant, only has Plcc and TAK1 to cooperate together (not providing).

Because PLC β 2 and TAK1 interact, the effect of PLC β 2 to activation TAK1 is examined.The overexpression of PLC β 2 demonstrates the phosphorylation (Fig. 3 G) of TAK1 in Thr187, Ser192 and Ser417 position significantly.After being activated, TAK1 phosphorylation IKK β or MKKs, cause the kinase whose activation (Adhikari of NF-к В, JNK and p38 of path label, A., M.Xu, and Z.J.Chen, 2007.Ubiquitin-mediatedactivation of TAK1and IKK.Oncogene.26:3214-3226).Further by the effect of reporter gene assay assessment PLC β 2 to activation NF-к В and AP-1.The expression of TAK1 and TAB1 in HEK293T cell significantly improves the reporter gene activity of NF-к В and AP-1, and the expression of PLC β 2 inhibits this to increase (not providing) with dose dependent fashion.For determining whether PLC β 2 affects the activity of TAK1 further, analyzing and being separated from PLC β 2 ^-/-the activity of the TAK1 of dsRNA induction in the scavenger cell of mouse.Stimulate, at PLC β 2 with poly (I:C) ^-/-in the scavenger cell of mouse than in the scavenger cell of wild-type mice, bring out the phosphorylation (Fig. 3 H) of TAK1 in Thr187, Ser192 and Ser417 position more.These data show the phosphorylation of PLC β 2 negative regulation TAK1.Result be consistent ground, when with poly (I:C) stimulate, PLC β 2 ^-/-scavenger cell show the higher activity (Fig. 3 I) of map kinase and NF-к В path than the scavenger cell of wild-type.These data show, PLC β 2 also inhibits the activity of the map kinase in downstream and the TAK1-mediation in NF-к В path.

The poly ubiquitination that the activation of TAK1 needs the K63-of TAK1 to connect, and the ubiquitination of TAK1 was regulated by the combining of protein TAB1 and TAB2 be connected with TAK1-.PLC β 2 significantly reduces the associating (Fig. 3 L) of TAB1 and TAK1 in dose-dependent mode.The poly ubiquitination that the K63-that TAB1 improves TAK1 connects, but the ubiquitination (Fig. 3 J) that the K63-that the overexpression of PLC β 2 significantly reduces TAK1 connects.On the contrary, the poly ubiquitination that the K63-of TAK1 that poly (I:C) induces connects is at PLC β 2 ^-/-scavenger cell in than being significantly enhanced in the scavenger cell of WT (Fig. 3 K).These results jointly show, PLC β 2 can target TAK1-TAB1 complex body, and the poly ubiquitination by suppressing the K63-of TAK1 to connect, the activity of negative regulation TAK1.

Active compound 2,4,6-trimethylammonium-N-(meta-3-trifluoromethyl-phenyl)-benzsulfamide (2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, m-3M3FBS) directly can stimulate the activity of Phospholipase C.Owing to identifying the negative regulation albumen of PLC β 2 as virus infection, the next effect of assessment m-3M3FBS in antiviral immunity reaction.The activity (Fig. 4 A and Fig. 4 B) in the phosphorylation of TAK1 and the map kinase in the scavenger cell of dsRNA-stimulation and NF-к В path significantly can be suppressed with m-3M3FBS process.Result is consistent ground, and the scavenger cell that m-3M3FBS is significantly reduced in poly (I:C)-stimulation comprises the expression of the pro-inflammatory cytokine of TNF (Fig. 4 C), IL-6 (Fig. 4 D) and IL-12 (Fig. 4 E).And, the clinical score (Fig. 4 G) of the mouse that CVA16-infects is reduced with m-3M3FBS process.M-3M3FBS also extends the survival (Fig. 4 H) of mouse that CVA16-infects, and reduces their histopathologies in lung (not providing) and muscle tissue (Fig. 4 F) and develop.These results show, m-3M3FBS can as the potential medicine for control CVA16 virus infection.

In the present invention, data show that PLC β 2 is highly brought out in the clinical sample of the scavenger cell infected from HFMD sufferer or CVA16-.The mouse lacked by the PLC β 2-of CVA16 infection has the more pro-inflammatory cytokine of a large amount and the virus titer of Geng Gao, and is more easily infected by the virus.These results show, the inhibitor that PLC β 2 can feed back as negative sense, to weaken the pro-inflammatory cytokine of the viral-induced generation of CVA16 in body.And, the histopathology development of CVA16-infecting mouse in lung and muscle tissue can be reduced with PLC activity agent m-3M3FBS process, and extend the survival of CVA-infecting mouse.Therefore, marking PLC β 2 is useful to treating such as HFMD communicate illness.

Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.

Claims (12)

1. whether authenticating compound regulates the method for Mammals TAK1 activity, it is characterized in that, comprises the steps:

Step 1, by mammalian cell compound incubation;

Step 2, the viral and virus antigen composition inductor stimulation by the cell of step 1;

Step 3, the TAK1 measured from the cell of step 2 are active;

Wherein, the pro-inflammatory cytokine that described TAK1 activity mark the pathogenic agent producing the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16 is brought out plays a crucial role.

2. method according to claim 1, is characterized in that, the mammalian cell of described step 1 is rodentine cell.

3. method according to claim 2, is characterized in that, described rodent is mouse.

4. method according to claim 1, is characterized in that, the mammalian cell of described step 1 is the cell of the mankind.

5. the method according to claim 1-4 any one, is characterized in that, described cell is selected from scavenger cell.

6. method according to claim 1, is characterized in that, described mammiferous cell is from cell culture.

7. method according to claim 1, is characterized in that, the virus of described step 2 and virus antigen composition be selected from poly I:poly C poly (I:C), H5N1, NDV, CAV16 any one or multiple.

8. method according to claim 1, is characterized in that, described step 3 is active from the TAK1 of the cell of step 2 by western blot test mensuration.

9. method according to claim 1, is characterized in that, the phospholipase Cβ 2 in described compound activating cell.

10. one kind is used for the treatment of the pharmaceutical composition of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16, it is characterized in that, described composition comprises albumen, polypeptide or their activating compounds with phospholipase Cβ 2 activity, and/or the salt that its physiology can tolerate, and pharmaceutically acceptable carrier or vehicle.

11. pharmaceutical compositions according to claim 10, is characterized in that, described compound is 2,4,6-trimethylammonium-N-(meta-3-trifluoromethyl-phenyl)-benzsulfamide.

12. compounds 2,4, the salt that 6-trimethylammonium-N-(meta-3-trifluoromethyl-phenyl)-benzsulfamide and/or its physiology can tolerate is for the preparation of the purposes of pharmaceutical composition, and this pharmaceutical composition is used for the treatment of the hand foot mouth disease caused by enterovirns type 71 or coxsackie virus A 16.