CN103804500B - Be used for the treatment of the polypeptide of chronic pain - Google Patents

Abstract

The invention discloses the polypeptide being used for the treatment of chronic pain, the motif of polypeptide is <b>ASADPGH</bGre atT.GreaT.GT, and motif has transmembrane transport signal segment by optional linking group coupling.Polypeptide of the present invention, both can Fyn regulates in inhibitory neuron NR2 phosphorylation, did not affect Fyn activity again, can treat chronic pain well.

Description

Be used for the treatment of the polypeptide of chronic pain

Technical field

The present invention relates to a peptide species, particularly a kind of polypeptide being used for the treatment of chronic pain.

Background technology

Chronic pain (is mainly divided into inflammatory pain, neurogenic pain) feature be hyperpathia (hyperalgesia), allodynia (allodynia), spontaneous pain (spontaneouspain) etc., it occurs all to relate to Synaptic plasticity and changes the central sensitization (centralsensitization) caused, for the generation of pain, cornu dorsale medullae spinalis (spinalcorddorsalhorn, SCDH) starting point of the travel of pain is described as, the cynapse signal transmission of SCDH can accept complicated, two-phase, and be the active neuroregulation relied on, under this adjustment, feeling to import into and accurately encoded and be passed into brain from peripheral nervous system.When periphery noxious stimulation have activated cornu dorsale medullae spinalis (spinalcorddorsalhorn by the Integrate adjustment (comprising immunocyte, spongiocyte etc. and neuronic dialogue etc.) of primary afferent neuron, SCDH) after Sensory neurone, to the depolarize of postsynaptic neuron tip be caused, activate NR on tip.Research shows, the excessive activation of NR is the indispensable important step that chronic pain central sensitization occurs.

NR is the ionotropic receptor of glutamate receptor, and be made up of function subunit (NMDAreceptor1, NR1) and adjustment subunit (NMDAreceptor2, NR2), NR2 divides NR2A, NR2B, NR2C and NR2D again.NR1 and a kind of NR2 may also have NR3 to form complete NR ionic channel.Under physiological conditions, after part is combined with NR, NR passage (being present in function subunit) is open, stream in positively charged ion, especially Ca ²⁺interior stream, this basic electrical activity (Basal activity) is that physiological function that NR is correlated with is rely the basis existed.As previously mentioned, the overactivity of NR plays a crucial role in the generation and maintenance of chronic pain central sensitization.

At present, dissimilar new anodyne just under development, take NR as target spot, and the excessive activation of managing suppression NR is the study hotspot of the DPNs such as chronic pain all the time.Suppress the activation of NR usually can use NR antagonist, but these antagonists all have neurotoxicity, the physiological function that NR relies on during the electrical activity of Antagonist block NR basis, can be destroyed.In a word, take NR as the basic electrical activity that first strategy of shot design disease therapy should not affect NR, reduce the interference to the physiological function that NR relies on.For NR passage, the architecture basics of the interior stream of positively charged ion is present in the function subunit of NR, and (activity of NR) the modulated subunit NR2 such as the intensity flowed in positively charged ion regulate, and therefore, regulate NR activity to be the New Policy for the treatment of chronic pain by regulation and control NR2.Contriver utilizes the reticent NR2B of SiRNA to alleviate the hyperpathia of pathology neurodynia rat in research before, and this has confirmed the feasibility taking NR2 as target treatment chronic pain.The tyrosine phosphorylation change of NR2 is the most basic mode that NR2 regulates NR activity, NR2 tyrosine phosphorylation, then NR increased activity, otherwise, dephosphorylation then makes NR activity reduce, therefore, the pathophysiological change that the tyrosine phosphorylation treatment NR overactivity of intervening NR2 causes may be avoid blocking NR, reduces the effective way to the physiological function interference that NR relies on.

NR2 tyrosine phosphorylation is regulated by the protein kinase family (Srcfamilyproteinkinases, SFKs) of the non-receptor-independent of neurone.Fyn is the SFKs member of being rich in spinal neuron, Fyn catalyzing N R2 tyrosine phosphorylation, is the signal joint that many signal paths regulate NR2 phosphorylation.Although multiple tyrosine residuess of the C-end of NR2A or NR2B can by SFKs family phosphorylation, but the phosphorylation in NR2BTyr1472 site regulates primarily of Fyn, some scholars think that NR2B is exactly the adjustment subunit that NR participates in pain, therefore, in SCDH postsynaptic Fyn regulate NR2 particularly the effect of NR2B tyrosine phosphorylation in chronic pain receive much concern.Many research shows, the hyperalgesic maintenance of chronic pain rat depends on the tyrosine phosphorylation in NR2BTyr1472 site that Fyn regulates, multi-signal molecule by Fyn catalyzing N R2 especially NR2B phosphorylation so that cause the generation of inflammatory pain or neuropathic pain.These results are pointed out, and the tyrosine phosphorylation of intervening Fyn regulates in neurone NR2 especially NR2B can treat chronic pain as inflammatory pain.Intervene Fyn in neurone and can use Fyn inhibitor to the tyrosine phosphorylation of NR2, but, this method poor specificity, because the catalysis region of the enzyme of homology family has conservative property, and Fyn is the tyrosine protein kinase being extensively present in Various Tissues in body, uses kinase inhibitor to suppress the activity of Fyn to affect the nerves and organize the physiological action of Fyn beyond system.From the angle that heredity is intervened, we also can suppress the expression of Fyn by gene therapy, also not can be applicable at present clinical have the high siRNA molecule vivo medicine-feeding system of high transfection efficiency, suitable transformation period, specificity and targeting.

Summary of the invention

The object of the present invention is to provide a kind of both can Fyn regulates in inhibitory neuron NR2 phosphorylation, do not affect again the polypeptide of Fyn activity.

Another object of the present invention is to provide a kind of preparation for the treatment of chronic pain.

The technical solution used in the present invention is:

Be used for the treatment of a polypeptide for chronic pain, its motif is ASADPGH(SEQIDNO:1), motif has transmembrane transport signal segment by optional linking group coupling.

Preferably, in the polypeptide of above-mentioned treatment chronic pain, the transmembrane transport signal segment of coupling is transmembrane transport peptide.

Preferably, above-mentioned transmembrane transport peptide is the fragment small peptide in Tat protein transduction domain.The sequence of transmembrane transport peptide is YGRKKRRQRRR(SEQIDNO:2).

Especially, the sequence being used for the treatment of the polypeptide of chronic pain is ASADPGH-YGRKKRRQRRR(SEQIDNO:3).

Treat a preparation for chronic pain, containing above-mentioned polypeptide.

The invention has the beneficial effects as follows:

Polypeptide of the present invention, both can Fyn regulates in inhibitory neuron NR2 phosphorylation, did not affect Fyn activity again, can treat chronic pain well.

Accompanying drawing explanation

Fig. 1 is the cure mechanism figure of the phosphorylated-activated and inflammatory pain of NR;

Fig. 2 is that Tat-FynP is to the effect diagram cultivating spinal cord neural cell NR2B tyrosine phosphorylated proteins level;

Fig. 3 is the effect diagram of different peptide to Fyn activity.

Embodiment

Be used for the treatment of a polypeptide for chronic pain, its motif is ASADPGH, and motif has transmembrane transport signal segment by optional linking group coupling.

The effect of transmembrane transport signal segment is to provide cell turn signal, will make cell that object peptide is transported to onset in cell.Preferably, transmembrane transport signal segment is transmembrane transport peptide.Preferably, above-mentioned transmembrane transport peptide is the fragment small peptide in Tat protein transduction domain.The sequence of transmembrane transport peptide is YGRKKRRQRRR.Certainly, other transmembrane transport signal segment also can be used, to promote that polypeptide carries out playing a role in cell.

Especially, the sequence being used for the treatment of the polypeptide of chronic pain is ASADPGH-YGRKKRRQRRR.

Treat a preparation for chronic pain, containing above-mentioned polypeptide.

Contriver place seminar finds under study for action, the combination of Fyn and NR2 is a kind of by PSD (postsynapticdensityprotein, PSD) indirect connection of albumen PSD-93 mediation, and PSD-93 only mediates the combination of Fyn and the NR2 of SFKs family.Fyn and PSD-95 combines and also mediates NR2 phosphorylation in the same way in fact.PSD-93/95 is a kind of special adaptin in kytoplasm, and its N-end contains PDZ1, PDZ2, PDZ3 tri-PDZ structural domains (with psD-95, dlg, and zo-1 albumen homology) be otherwise known as PDZ domain protein.As the Major Members of PDZ domain protein, PSD-93/95 is combined by PDZ2 structural domain and NR.Fyn is a kind of protein kinase of non-receptor-independent, it is by the direct combination of the PDZ3 structural domain of the SH2 structural domain beyond the enzyme zone of action and PSD-93/95 thus form complex mediated NR phosphorylation with NR, this combination is the physical bond mode of the uniqueness not relying on tyrosine phosphorylation degree, the Tyr phosphorylation (Tyr420) of FynC end Tyr dephosphorylation and kinase domain can make Fyn activate, contrary with the phosphorylation that acceptor lowered by the Phosphoric acid esterase that it has been generally acknowledged that, tyrosine phosphatase α makes Fyn dephosphorylation in cynapse promote the formation of this mixture on the contrary, and then raise NR2 tyrosine phosphorylation.When chronic pain is as inflammatory pain, many signaling molecules indirectly or can directly act on Fyn and promote NR2 phosphorylation.

In a word, PSD-93/95 links together with NR all the time, and when stimulus signal imports into, the PDZ3 structural domain of PSD-93/95 is combined with FynSH2 structural domain again, makes Fyn, PSD-93/95, NR trail together, causes NR phosphorylated-activated (Figure 1A).Accordingly, contriver imagines, can by suppressing the interaction of FynSH2 and PSD-93/95PDZ3, the trail of Fyn and PSD93/95 and NR can be suppressed, thus suppression NR's is phosphorylated-activated, prevents or reverses central sensitization, reaching the therapeutic action (Figure 1B) to inflammatory pain.This strategy is based on the strategy suppressing FynSH2 structural domain and PDZ3 domain interaction, do not affect the integrity of NR passage, retain the basic electrical activity of NR, therefore the physiological function interference relied on NR is little or noiseless, does not affect the effect of Fyn to the phosphorylated regulation of other albumen beyond neural system NR2 and other histoorgan Fyn.

Below in conjunction with experiment and experimental data, further illustrate the present invention.

The concrete sequence of the Tat-FynP used below in experiment is ASADPGH-YGRKKRRQRRR.

Tat-FynP is on the impact of cultivating spinal cord neural cell NR2B tyrosine phosphorylated proteins level

Tat-FynP, FynP, mTat-FynP add the culturing cell of hatching with Fyn agonist in advance and the culturing cell not adding agonist respectively, hatch rear cell pyrolysis liquid routinely step extract albumen and divide two parts (another points standby NR2B total protein levels detect), utilize specificity p-NR2B antibody, observed the change of each group of tyrosine phosphorylation NR2B protein level by Western-blot.

40 μ g total protein loadings, two pieces of parallel glue on every sample, to determine molecular weight of albumen and loading consistence, after SDS-PAGE electrophoretic separation, with wet robin electrotransfer on pvdf membrane, condition is 150mA2 hour, transfering buffering liquid: 25mmol/LTris, 190mmol/L glycine, 0.05%SDS, 20% methyl alcohol, after transfer, immerses confining liquid by pvdf membrane, 4 DEG C are spent the night, the BufferA(10mmol/LTris of confining liquid composition: 1%BSA, 100mmol/LNaCl, 0.1%Tween20).Add primary antibodie (the 1:1000 goat-anti p-NR2B with confining liquid dilution, be SantaCruz company), BufferA washes film 10 minutes × 3 times, add two anti-(the biotin labeled Ma Kangyang of 1:300 or goat anti-rabbit iggs, Beijing Zhong Shan), hatch 30 minutes for 37 DEG C, wash film 3 times, add 3 anti-(1:300 horseradish enzyme labelling chain enzyme avidin, Beijing Zhong Shan), BufferA washes film 3 times, finally film is put into DAB solution to develop the color, once the color depth of protein band reaches requirement, namely use ddH2O rinsing, termination reaction, takes film photo afterwards.By the analyzing and processing on GelDoc2000 gel imaging system of the target protein band on pvdf membrane, phosphorylation NR2B protein content is with ODu × cartographic represenation of area.

Experimental result as shown in Figure 2.These data show peptide T at-FynP of the present invention, can Fyn regulates in inhibitory neuron NR2 (being NR2B in this experiment) phosphorylation.

Tat-FynP is on the impact (verifying its specific action or targeting) of Fyn kinase activity

By radioimmunoassay technical measurement Fyn kinase activity.

The spinal cord neural cell Fyn kinase activity change that Tat-FynP, FynP, mTat-FynP hatch is measured respectively with radioimmunoassay technology test kit.The dosage of Tat-FynP adopts 10 times that can suppress the interactional amount of Fyn and PSD-95 respectively.

Extracted the cell protein of hatching by sonioation method, measure protein concentration by lory method.Kinase activity detects: add 20 μ lproteinA/GPlusAgarose, 4 DEG C of horizontal shaker 100rpm30min, 7000rpm centrifuging and taking supernatant liquor.Fyn antibody 4 DEG C of shaking tables are added in 500 μ g albumen, centrifugal, buffer washing precipitation, the resuspended precipitation of bufferA after above-mentioned steps 3 times.Build [γ- ³²p] ATP reaction system, 10 μ l reaction systems are added mixed solution and hatches 20min, SDS-PAGE electrophoresis after termination reaction, is transferred on nitrocellulose filter, 2h ,-70 DEG C, compressing tablet 48-72h in dark surrounds, development, fixing.Image is through data system analysis process.Result as shown in Figure 3.Each group of Fyn is active in significant difference, polypeptide of the present invention is described, does not affect Fyn activity.

Tat-FynP intrathecal injection is in advance on the impact of trace formaldehyde inflammatory pain rat model pain behavior

Animal is divided into normal group, inflammatory pain group (CFA), inflammatory pain group again sub-model make before intrathecal injection Tat-FynP, FynP, mTat-FynP group in advance, measure rat MWT and TWD respectively at 1d, 3d, 4d and 5d before left hind subplantar injection trace formaldehyde and after injection.Found that Tat-FynP intrathecal injection pre-treatment has therapeutic action to inflammatory pain rat.

The two kinds of equal not statistically significant of Pain behaviour Indexes Comparison differences (P>0.05) of each group rat before modeling; Compare with normal rat group, 3d after CFA group 4d(is equivalent to intrathecal injection in treatment medicine) namely can be observed the MWT value decline highly significant of suffering from foot, TWD value obviously extends, have statistical significance (P<0.01) with basic value before model and NS group comparing difference, and this variation tendency continues to 5d after intrathecal injection.The ascensional range of fall and TWD value that Tat-FynP group can make rat suffer from sufficient MWT value reduces, compare with CFA group that there were significant differences (P<0.01), and the comparing without significant difference (P>0.05) with CFA group of FynP group and mTat-FynP group rat MWT and TWD value.(table 1a, table 1b).

Before table 1a, the modeling of each group rat and after intrathecal injection or the change of corresponding time point MWT

a:P<0.01vsNS，b:P<0.01vsCFA。

The change of TWD after table 1b, the modeling of each group rat

a:P<0.01vsNS，b:P<0.01vsCFA。

<110> Guangzhou General Hospital Guangzhou Military Command

<120> is used for the treatment of the polypeptide of chronic pain

<130>

<160>3

<170>PatentInversion3.5

<210>1

<211>7

<212>PRT

<213> artificial polypeptide

<400>1

AlaSerAlaAspProGlyHis

15

<210>2

<211>11

<212>PRT

<213> artificial polypeptide

<400>2

TyrGlyArgLysLysArgArgGlnArgArgArg

1510

<210>3

<211>18

<212>PRT

<213> artificial polypeptide

<400>3

AlaSerAlaAspProGlyHisTyrGlyArgLysLysArgArgGlnArg

151015

ArgArg

Claims (4)

1. be used for the treatment of a polypeptide for chronic pain, its motif is aSADPGH, motif coupling is classified as in order yGRKKRRQRRRtransmembrane transport peptide.

2. polypeptide according to claim 1, is characterized in that: motif is classified as in order by linking group coupling yGRKKRRQRRRtransmembrane transport peptide.

3. polypeptide according to claim 1, is characterized in that: the sequence of described polypeptide is aSADPGH-YGRKKRRQRRR.

4. treat a preparation for chronic pain, it is characterized in that: containing the polypeptide described in claim 1,2 or 3 in described preparation.