CN1569889A - Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use - Google Patents
Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use Download PDFInfo
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- CN1569889A CN1569889A CNA031502059A CN03150205A CN1569889A CN 1569889 A CN1569889 A CN 1569889A CN A031502059 A CNA031502059 A CN A031502059A CN 03150205 A CN03150205 A CN 03150205A CN 1569889 A CN1569889 A CN 1569889A
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- residue
- type
- rnaiii
- bacterium
- rap
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention relates to a group of cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus, Steroidal C#-[1]-X#-[1]-H#-[1]-A#-[2]-H#-[1]-A#-[2]-C#-[1], wherein C is natural L-type aminothiopropionic acid residue or its D-type isomer, X is any one of the four natural L-type amino acid residue or its D-type isomers, i.e. glutamine residue (Q), glutacid residue (E), aspartate residue (D), asparagines residue (N), H is L-type histidine residue or its D-type isomer, A is natural L-type aromatic residue or its D-type isomer. The polypeptides can be applied in preparing staphylococcus aureus resistant medicament.
Description
Technical field
The present invention relates to one group of cyclic polypeptide, be specifically related to RNAIII activator bonded cyclic polypeptide enough and golden Portugal bacterium autocrine, this toxin in conjunction with the golden Portugal of the special inhibition of Toplink bacterium produces.The invention still further relates to the application of these cyclic polypeptides in field of medicaments.
Background technology
Streptococcus aureus (golden Portugal bacterium) is the common Gram-positive pathogenic bacterium of a class, is one of major microorganisms that causes mortality diseases such as burn and war wound infection, pneumonia, endocarditis, septicemia, toxic shock.The number of annual only nosocomial infection gold Portugal bacterium just surpasses millions of.Clinically adopt of the treatment of golden Portugal bacterium united the antibiotic way of use more at present, but effect is unsatisfactory.Because golden Portugal bacterium very easily produces resistance and do not have good solution, many microbiotic commonly used are invalid to it, and controlling the bacterium infection of golden Portugal is one of clinical medicine problem demanding prompt solution.
The main morbid substance of gold Portugal bacterium is a toxin, comprises hemolytic toxin, leueocidin, enterotoxin etc.Current research shows that the synthetic of golden Portugal these virulence factors of bacterium is to be subjected to a kind of RNA of adjusting molecule, and RNAIII control.RNAIII activates the genetic transcription of virulence factor, regulates the translation of virulence factor by base complementrity.Low in early stage its RNAIII level of the logarithm of bacterial growth, but can increase by 40 times to logarithm RNAIII in late period level, and the level of RNAIII is by golden Portugal bacterium self excretory albumen, RANIII activator (RNAIII activating protein), also claim what RAP regulated, so factor R AP is called staphylococcus aureus virulence stimulating factor again.The gold bacterium continuous release RAP of Portugal just has the effect that virulence factor produces that activates after RAP reaches finite concentration.The golden Portugal bacterium itself that does not have RAP to produce is also not pathogenic.Balaban in 1998 etc. deliver result of study at " Science " magazine and show; RAP immune animal with their preparation; its antibody can protect mouse to avoid infection (the Balaban N of golden Portugal bacterium effectively; et al.Autoinducer ofvirulence as a target for vaccine and therapy against Staphylococcus aureus.Science; 1998,280 (17): 438-440).At present, existence and the sequence of relevant RAP still have arguement, also do not see bibliographical information.
Summary of the invention
The purpose of this invention is to provide one group can with the RNAIII activator bonded cyclic polypeptide of golden Portugal bacterium autocrine, this produces in conjunction with toxin of the golden Portugal of the special inhibition of Toplink bacterium.
Another object of the present invention provides the application of these cyclic polypeptides in field of medicaments.
For realizing first purpose of the present invention, we isolate the active albumen of a kind of RNAIII of inhibition with improved literature method from China's clinical pathogenic golden Portugal bacteria strain 04018, and we are with it called after RAP (Y).We adopt display technique of bacteriophage to filter out from the random loops peptide library can specially combine and suppress its active micromolecule polypeptide with RAP (Y) molecule, purpose is the inducing action that blocking-up RAP (Y) contratoxin produces, thereby cut off the signal transduction that golden Portugal verticillium toxin produces, infect the new approach of opening up for treating golden Portugal bacterium.No matter polypeptide of the present invention does not see any bibliographical information from originating or being new fully on structure, its effect target protein RAP (Y) does not see bibliographical information yet.
Of the present invention can with the RNAIII activator bonded cyclic polypeptide of golden Portugal bacterium autocrine, be polypeptide with following formula:
C
1-X
1-H
1-A
2-H
1-A
2-C
1。
Wherein, C represents natural L-type cysteine residues or its D-type isomer; X represents any one in following four kinds of natural L-type amino-acid residues or its D-type isomer, i.e. glutamine residue (Q), glutaminic acid residue (E), asparagicacid residue (D) and asparagine residue (N); H represents histidine residues or its D-type isomer; A represents natural L-type die aromatischen Aminosaeuren residue or its D-type isomer, as tryptophan residue (W), tyrosine residues (Y) or phenylalanine residue (F) etc.; The number of footnote digitized representation amino-acid residue.
Have said structure polypeptide can with staphylococcus aureus virulence stimulating factor RAP (Y) specific combination, and suppress its activity.Be embodied in, no matter at external polypeptide with said structure pattern is as being illustrated on the phage or external chemosynthesis or recombinant expressed with gene engineering method with which kind of form, can both with RAP (Y) specific combination, and suppress in the golden Portugal bacterium expression by the RNAIII of RAP (Y) regulation and control, experimentation on animals is the result show, the polypeptide with said structure pattern can suppress the microbial infection in golden Portugal.
Micromolecule polypeptide of the present invention can make by chemosynthesis of the prior art or with the recombinant expressed method of gene engineering method.
Bacterium produced the inducible enzyme that decomposes effective group in the microbiotic after the reason that the tradition antibiotic therapy develops immunity to drugs was mainly medication under survival pressure.The scheme that the treatment gold Portugal bacterium that the present invention utilizes the active polypeptide of special inhibition RAP (Y) to set up infects, owing to kill bacteria not makes the pathogenic forfeiture of bacterium, so can not resemble traditional antibiotic therapy bacterium is not produced selective pressure and produces drug resistance strain, this just infects for perplex clinical resistance gold Portugal bacterium always that this is common, multiple and have fatefulue treatment of diseases to find new outlet.
Description of drawings
Fig. 1 is a purifying rap protein electrophoretogram
Fig. 2 is the active detection figure of RAP (Y)
Fig. 3 combines situation for the ELISA method detects the GST-C7 fusion rotein with RAP (Y)
Fig. 4 is the influence of RAP (Y) binding peptide-gst fusion protein to RNAIII level in the golden Portugal mycetocyte
The present invention is significant to the micromolecule polypeptide medicine that development novel anti gold Portugal bacterium infects, and is with a wide range of applications and vast market prospect, thereby has realized second purpose of the present invention.In addition, staphylococcus aureus virulence stimulating factor RAP (Y) binding peptide also has the potential using value as biotechnological formulation, as can be used for the affinity purification of RAP (Y) or be used for detection of RAP (Y) etc. as probe.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
The separation and purification of embodiment 1.RAP (Y)
To grow to logarithm late period (4.7 * 10
9The golden bacterium bacterium liquid 7000 * g of Portugal of bacterium/ml) got supernatant in centrifugal 15 minutes, boiled after 10 minutes centrifugal 15 minutes, got supernatant again.After concentrating ten times with the filter membrane volume of molecular weight cut-off 10kD, get 3ml with the separation and purification of molecular sieve layer analysis method.Separating medium be S-300 (100ml, 2.2 * 26cm), damping fluid is 50mMTris-Cl, pH=8.0 collects the target protein peak.Conventional SDS-PAGE polyacrylamide detected through gel electrophoresis purity and observed molecular weight.The result shows that the albumen that is obtained is single band, molecular weight be about 38kD (see accompanying drawing 1, among the figure 1 the expression rap protein, 2 the expression protein standards, 3 the expression thrombin of beef (～36kD).
Ordinary method is extracted total RNA of golden Portugal bacterium, behind the denaturing formaldehyde electrophoresis, by half-dried transfer RNA is transferred to nylon membrane.60 ℃ of prehybridizations after 1 hour, add the special dna probe of labeled rna III.60 ℃, after 16 hours, wash film and carry out radioautograph.In the active detection of RAP (Y), positive control adds 4.5ml culture medium inoculated logarithmic growth early stage (5 * 10 for concentrated 10 times logarithmic growth supernatant in late period 0.5ml (containing high density RAP (Y))
8Cultivated 60 minutes by bacterium the golden Portugal of bacterium/ml); Sample is inoculated same golden Portugal bacterium cultivation 60 minutes for the albumen that the 5ml substratum adds the 0.5mg purifying; Negative control is that the same golden Portugal of 5ml culture medium inoculated bacterium was cultivated 60 minutes.
1.Northern blot result shows: the albumen of purifying RNAIII in the early stage activating cells of logarithmic growth transcribes, and its level is significantly raise (see accompanying drawing 2,1 represents to concentrate supernatant among the figure, 2 expression purifying RAP (Y), 3 expression substratum contrasts).The 38kD albumen RAP (Y) that shows purifying has the biological action that RNAIII transcribes in the golden Portugal of the activation mycetocyte.
The screening of embodiment 3.RAP (Y) binding peptide
RAP (Y) the albumen bag quilt of at first using 100 μ l purifying is put 4 ℃ and is spent the night in elisa plate.Behind 2% gelatin sealing 1h, add phage peptide library, incubated at room 1h, with TBST (50mmol/LTris-HCl, 0.1%TWEEN20, pH7.5) phage of the non-specific combination of washing is used the phage of 0.2mmol/L glycine-HCl pH2.2 wash-out specific combination again, and elutriant is with 1mmol/L Tris-HCl pH9.0 neutralization.The method that provides according to test kit is measured the titre of the phage in the elutriant, with the titre of the wash-out bacteriophage of the target protein that do not wrap quilt in contrast, measures input-output ratio.Simultaneously with wash-out with RAP bonded phage amplification, and measure its titre down, be used for the screening of next round.After taking turns screening through 3, the input-output of mensuration are significantly improved, and productive rate is by 0.865 * 10 of the first round
-4% brings up to 3.846 * 10
-2% promptly specially during third round has obtained great enrichment with RAP (Y) bonded phage clone.18 clones of picking check order at random.Obtain having the polypeptide of following formula after the analysis:
C
1-X
1-H
1-A
2-H
1-A
2-C
1。
Wherein, C represents natural L-type cysteine residues or its D-type isomer, X represent following four kinds of natural L-type amino-acid residues or its D-type isomer any one, be glutamine residue (Q), glutaminic acid residue (E), asparagicacid residue (D) or asparagine residue (N), H represents histidine residues or its D-type isomer, A represents natural L-type die aromatischen Aminosaeuren residue or its D-type isomer, as tryptophan residue (W), tyrosine residues (Y) or phenylalanine residue (F) etc., the number of footnote digitized representation amino-acid residue has the polypeptide of said structure general formula.
The gene engineering expression of embodiment 4.RAP (Y) binding peptide-gst fusion protein and
Influence to RNAIII level in the golden Portugal mycetocyte
Utilize molecular biology method to make up GST-ring 7RAP (Y) binding peptide antigen-4 fusion protein gene expression vector, after order-checking is identified correctly, with the GST-C7 fusion rotein at expression in escherichia coli, method by ELISA detects, the GST-C7 fusion rotein of finding separation and purification can combine with RAP (Y) specificity, as accompanying drawing 3.
Utilize embodiment 2 measuring method for activity to find, GST-C7 fusion rotein (0.2mg/ml) can obviously suppress RNAIII level in the golden Portugal bacterium, and (see accompanying drawing 4,1 expression substratum contrasts among the figure and the GST reference protein does not have this effect, 2 expression GST contrasts, 3 expression GST-C7 fusion roteins).
Claims (3)
1. can combine with the RNAIII activator of golden Portugal bacterium autocrine, and have the cyclic polypeptide of following structure:
C
1-X
1-H
1-A
2-H
1-A
2-C
1
In the formula, C represents natural L-type cysteine residues or its D-type isomer; X represent following four kinds of natural L-type amino-acid residues or its D-type isomer any one: glutamine residue (Q), glutaminic acid residue (E), asparagicacid residue (D) and asparagine residue (N); H represents L-type histidine residues or its D-type isomer; A represents natural L-type die aromatischen Aminosaeuren residue or its D-type isomer; The number of footnote digitized representation amino-acid residue.
2. the described cyclic polypeptide of claim 1 is characterized in that A represents tryptophan residue (W), tyrosine residues (Y) or phenylalanine residue (F).
3. claim 1 or the 2 described cyclic polypeptides application in the anti-golden Portugal of preparation bacterium infection medicine.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031502059A CN1569889A (en) | 2003-07-21 | 2003-07-21 | Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use |
| PCT/CN2003/000831 WO2005007685A1 (en) | 2003-07-21 | 2003-09-28 | Cylic polypeptides that can combine with rnaiii activating protein secreted by staphylococcus aureus and their medical use |
| AU2003271038A AU2003271038A1 (en) | 2003-07-21 | 2003-09-28 | Cylic polypeptides that can combine with rnaiii activating protein secreted by staphylococcus aureus and their medical use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031502059A CN1569889A (en) | 2003-07-21 | 2003-07-21 | Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1569889A true CN1569889A (en) | 2005-01-26 |
Family
ID=34069993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031502059A Pending CN1569889A (en) | 2003-07-21 | 2003-07-21 | Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1569889A (en) |
| AU (1) | AU2003271038A1 (en) |
| WO (1) | WO2005007685A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101791397A (en) * | 2010-03-17 | 2010-08-04 | 中国人民解放军军事医学科学院基础医学研究所 | Application of RNase III protein in staphylococcus aureus for preventing and treating staphylococcus aureus infection |
| WO2017190619A1 (en) * | 2016-05-03 | 2017-11-09 | 重程投资管理（上海）有限公司 | Chemosynthetic cyclo-heptamodified peptide capable of inhibiting toxin of staphylococcus aureus and use thereof |
| WO2024037263A1 (en) * | 2022-08-16 | 2024-02-22 | 重程投资管理（上海）有限公司 | Synthetic peptide having low toxicity in vivo for inhibiting generation of toxins of staphylococcus aureus and use thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6291431B1 (en) * | 1997-12-19 | 2001-09-18 | Panorama Research | Methods and compositions for the treatment and prevention of Staphylococcal infections |
-
2003
- 2003-07-21 CN CNA031502059A patent/CN1569889A/en active Pending
- 2003-09-28 WO PCT/CN2003/000831 patent/WO2005007685A1/en active Application Filing
- 2003-09-28 AU AU2003271038A patent/AU2003271038A1/en not_active Abandoned
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101791397A (en) * | 2010-03-17 | 2010-08-04 | 中国人民解放军军事医学科学院基础医学研究所 | Application of RNase III protein in staphylococcus aureus for preventing and treating staphylococcus aureus infection |
| WO2017190619A1 (en) * | 2016-05-03 | 2017-11-09 | 重程投资管理（上海）有限公司 | Chemosynthetic cyclo-heptamodified peptide capable of inhibiting toxin of staphylococcus aureus and use thereof |
| CN109071604A (en) * | 2016-05-03 | 2018-12-21 | 重程投资管理（上海）有限公司 | A kind of seven modified peptides of chemical synthesis ring that can inhibit S. aureus L-forms toxin and its application |
| US10905735B2 (en) | 2016-05-03 | 2021-02-02 | Zhongcheng Investment Management (Shanghai) Co., Ltd | Chemosynthetic cyclo-hepta modified peptide capable of inhibiting toxin of Staphylococcus aureus and use thereof |
| CN109071604B (en) * | 2016-05-03 | 2022-02-08 | 重程投资管理（上海）有限公司 | Chemically synthesized cyclic hepta-modified peptide capable of inhibiting staphylococcus aureus toxin and application thereof |
| WO2024037263A1 (en) * | 2022-08-16 | 2024-02-22 | 重程投资管理（上海）有限公司 | Synthetic peptide having low toxicity in vivo for inhibiting generation of toxins of staphylococcus aureus and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003271038A1 (en) | 2005-02-04 |
| WO2005007685A1 (en) | 2005-01-27 |
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