One group of novel HRP5 analogue and preparation method thereof
Technical field
The invention belongs to field of medicaments, in particular to one group, novel there is HRP5 analogue of broad spectrum antibiotic activity and preparation method thereof.
Background technology
Since penicillin is found, microbiotic is the powerful mean of human treatment's cause pathogeny imcrobe infection always, but along with the antibiotic abuse of tradition, increasing pathogenic bacteria starts traditional microbiotic to produce resistance, so be badly in need of the brand-new antibacterials of searching-class, comes substitute antibiotics to use.
Cationic antibacterial peptide (cationic antimicrobial peptides) is general cationic (being rich in arginine and Methionin) polypeptide being comprised of 12-50 amino-acid residue that plant and animal produces, and can protect host not to be subject to extraneous cause pathogeny imcrobe infection.The antimicrobial spectrum of cationic antibacterial peptide is wider than traditional microbiotic, not only gram-positive microorganism and Gram-negative bacteria is had to anti-microbial effect, but also have antimycotic and antiviral activity from insect, pig, the frog and people's cationic antibacterial peptide.Tradition microbiotic passes through to eliminate microorganism growth or existence essential condition, as makes enzyme denaturation, reach the object of sterilization, but bacterium just can be resisted this type of antibiotic attack after needing only a kind of transgenation.During cationic antibacterial peptide passes through and method and the bacterial cell membrane interaction of electric charge, with this, penetrate and kill bacterium, greatly reduced the possibility of bacterium generation resistance.But natural cationic antibacterial peptide is not flawless, part antibacterial peptide has certain toxicity to eukaryote, and cause of disease object height is killed active time and is often accompanied by Eukaryotic hemolytic action.Therefore how to improve that it is active and at utmost to reduce its toxicity be the difficult point of current antimicrobial peptide medicaments exploitation and wish place.Current research is mainly using the both sexes antibacterial peptide of alpha-helix as research object, around cationic amino acid and hydrophobic amino acid quantity and position, antibacterial peptide is carried out to structure of modification, as residue, replace, intercept the partial sequence of natural antibacterial peptide and the positive charge content of increase peptide chain etc., obtained greater advance.
Histatins (histidine rich protein, HRPs or histatins) is-class is present in the cationic polypeptide that is rich in Histidine in primate Parotid and submandibular gland secretory product.Now isolated at least 12 kinds of people HRPs (HRP1-12), wherein HRP1, HRP3 and HRP5 are main histatinses, account for the 85%-90% of total HRPs, and respectively by 38,32,24 amino acid form.HRP1 and HRP3 be by different genes encodings, HRP5 be HRP3 through the product of protease hydrolysis, be included in 24 residues of N-terminal of HRP3.Coding HRP1,3 gene has been sequenced and has been positioned on No. 4 karyomit(e) q13 of the mankind.Now there are some researches show: HRPs has multiple active biological function, and its anti-microbial effect is particularly important, as suppressed, kill streptococcus mutans and Candida albicans etc.
HRP5 contains 24 amino-acid residues, and molecular weight is 3037D.Its aminoacid sequence is Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr (SEQ ID NO:1).The scholars such as Raj think that the characteristic of HRP5 anti-candida albicans is relevant with its aminoacid sequence, they respectively by HRP5 and residue segment N16 (1-16 amino acids), C16 (9-24 amino acids), C14 (11-24 amino acids), C12 (13-24 amino acids), C10 (15-24 amino acids) and M10 (7-16 amino acids) respectively with the Candida albicans effect of different concns, detect the ability of its anti-Candida albicans.Test-results shows that C16 and C14 have the ability of stronger inhibition albicans growth, and the activity of the anti-Candida albicans of HRP5 and C16 is basically identical, and the activity that contains equally 16 amino acid whose N16 anti-candida albicanses is well below C16.The functional zone of the anti-candida albicans of this results suggest HRP5 are positioned at carboxyl terminal.For the further relation of the structure and function of research HRP5, many scholars are replaced and have been obtained a series of varient by residue, wherein more importantly dhvar1, dhvar2, dhvar4, dhvar5.They are HRP5 active zone (11-24 amino acids) varients.
Summary of the invention
Polypeptide or its pharmaceutical salts, described polypeptide has following aminoacid sequence:
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Leu-Xaa11-Lys-Tyr-Leu-Arg-Xaa16-Xaa17-Xaa18(SEQ?ID?NO:2)
Wherein Xaa1 is Cys or disappearance;
Xaa2 is Lys, D-Lys, Arg, D-Arg, HoR or disappearance;
Xaa3 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe, Thi or disappearance;
Xaa4 is Lys, D-Lys, Arg, D-Arg, Aib or HoR;
Xaa5 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa6 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa7 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa8 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa9 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa11 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa16 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa17 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa18 is Cys or disappearance, and when Xaa1 and Xaa18 are all Cys, this polypeptide can form cyclic peptide.
As a preferred embodiment of the present invention Xaa1 and Xaa18, be all Cys or lack simultaneously; The preferred Arg of Xaa2, D-Arg or disappearance; The preferred Phe of Xaa3, D-Phe or disappearance; The preferred Lys of Xaa4, D-Lys, Arg, Aib or HoR; The preferred Lys of Xaa5, Arg or D-Arg; The preferred Leu of Xaa6, Ile or Nle; The preferred Trp of Xaa7, Phe, D-Phe or Thi; The preferred Lys of Xaa8, D-Lys or Arg; The preferred Lys of Xaa9, D-Lys or Arg; The preferred Leu of Xaa11, D-Phe or Phe; The preferred Lys of Xaa16, D-Lys or Arg; The preferred Trp of Xaa17, Phe, D-Phe or Thi.
Amino-acid residue involved in the present invention had both comprised natural amino acid, also comprised alpha-non-natural amino acid.The corresponding trigram code table of natural amino acid involved in the present invention is as shown in table 1, and the corresponding trigram code table of alpha-non-natural amino acid involved in the present invention and structure are as shown in table 2.
If all amino-acid residues involved in the present invention, without particular limitation of its configuration, represent L-type amino acid.
Polypeptide involved in the present invention, can be both linear peptides, can be also cyclic peptide.When Xaa1 and Xaa18 are all Cys, can be by adding oxygenant to make to form disulfide linkage between Xaa1 and Xaa18, now this peptide becomes cyclic peptide by linear peptides.
When polypeptide involved in the present invention is linear peptides, its C-terminal can be both the form of carboxylic acid, can be also the form of acid amides, and preferably the form with acid amides exists.
Table 1: natural amino acid trigram code table
Table 2: alpha-non-natural amino acid trigram code table and structure
The polypeptide (AMP-1) that discloses a kind of linearity in the preferred embodiment of the present invention, its aminoacid sequence is: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ ID NO:3)
Another discloses the polypeptide (AMP-2 of one group of linearity the present invention preferred embodiment, AMP-3, AMP-4, AMP-5, AMP-6, AMP-7, AMP-8, AMP-9, AMP-10, AMP-11, AMP-12), its aminoacid sequence is respectively: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH
2(SEQ ID NO:4) D-Lys-D-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Ly s-D-Phe-NH
2(SEQ ID NO:5) D-Lys-D-Arg-Leu-Phe-D-Lys-D-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH
2(SEQ IDNO:6)
Phe-Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ?ID?NO:7)
Lys-Arg-Leu-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQ?ID?NO:8)
Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQ?ID?NO:9)
Arg-Leu-Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQID?NO:10)
Lys-Arg-Leu-Thi-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO:11)
Lys-Arg-Nle-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO:12)
Aib-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO:13)
HoR-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO:14)
Of the present invention another preferred embodiment discloses a kind of polypeptide (AMP-13) of ring-type, and its aminoacid sequence is:
The present invention also has an object to be just to provide the preparation method of above-mentioned linearity and ring type polypeptide, can adopt the familiar solid state chemistry synthetic technology of those skilled in the art to prepare linear peptides and on the basis of linear peptides, prepare cyclic peptide by hydrogen peroxide oxidation method.
The preparation process of solid state chemistry synthetic technology is as follows:
(1) solid-phase synthetic peptide on resin;
(2) product of step (1) is carried out to cracking in trifluoroacetic acid or hydrofluoric acid, preferred trifluoroacetic acid, and add Side chain protective group scavenging agent, then with the ice ether sedimentation polypeptide of 5-20 times of volume, centrifugal, abandon supernatant, then precipitate 4-5 time with ice ether repetitive scrubbing, vacuum-drying, obtains thick peptide;
If peptide C end involved in the present invention be carboxylic acid form step (1) adopt Wang resin to synthesize; If peptide C end involved in the present invention be acid amides form step (1) adopt Rink Amide mbha resin to synthesize.
Step (1) is to carry out in liquid phase environment, specifically comprises: soak resin-remove amino protecting group-washing-coupling amino acid-monitor-wash-remove amino protecting group (sequentially coupling remaining amino acid)-dry resin.
Wherein amino protecting group refers to the chemical group of introducing for the amino of protection participation condensation reaction.Described amino protecting group is selected from: tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Z) and 9-fluorenyl-methyl carbonyl (Fmoc), preferably 9-fluorenyl-methyl carbonyl (Fmoc).
As an advantage of solid-phase polypeptide synthetic technology, to the side chain of partial amino-acid, can protect by introducing chemical group, for example Arg and HoR can adopt pentamethyl-benzo furans-5-alkylsulfonyl (Pbf); Cys can adopt trityl (Trt); Lys and Trp can adopt tertbutyloxycarbonyl (Boc); Tyr can adopt the tertiary butyl (tBu).Described blocking group is not limited to this, can carry out choose reasonable according to this area conventional scheme.
The liquid phase environment solvent for use of step (1) is selected from: dimethyl formamide (DMF) or methylene dichloride (DCM), preferably DMF.
In step (1), remove the agent that removes that amino protecting group need to add amino protecting group, piperidines (PIP) solution is selected in the agent that removes of amino protecting group, concentration 10-40% (PIP/DMF), and the time of removing is 20-50min.Preferred concentration is 20-25% (PIP/DMF), removes time 25-35min.
In step (1), amino acid whose coupling need to add coupling reagent, coupling reagent by: carbodiimide type reagent or benzotriazole salt type reagent and I-hydroxybenzotriazole (HOBt) form.
Carbodiimide type reagent comprises dicyclohexylcarbodiimide (DCC), DIC (DIC) and N-diamino propyl group-N-ethyl carbodiimide (EDC).
Benzotriazole salt type reagent comprises 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), O-benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HBTU), phosphofluoric acid benzotriazole-1-oxygen base three (dimethylamino) phosphorus (BOP), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP) etc.
The preferred DIC of coupling reagent (DIC) and I-hydroxybenzotriazole (HOBt), 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester (TBTU) and I-hydroxybenzotriazole (HOBt), most preferably DIC (DIC) and I-hydroxybenzotriazole (HOBt).
" monitoring " in step (1) adopts the condensation reaction of triketohydrindene hydrate detection method monitoring polypeptide.
The amino acid that is linked in sequence in step (1) refers to according to polypeptid acid sequence and to N end, connects one by one amino acid from C end.
The described Side chain protective group scavenging agent of step (2) be selected from as thioanisole, tri isopropyl silane, phenol, water, 1,2-ethandithiol, meta-cresol etc. two or more combination and prepare and obtain by 5-20% (V/V) with trifluoroacetic acid or hydrofluoric acid.Preferred trifluoroacetic acid (TFA): thioanisole: 1,2-ethandithiol: 75% phenol: water=85: 5: 5: 4: 1.
Useful is especially for meeting the specification of quality of medicinal use, and polypeptide preparation method provided by the present invention also comprises that employing conventional means carries out purifying.The purification process adopting can be reverse-phase chromatography or ion exchange chromatography, preferably reverse-phase chromatography.
The antibacterial activity in vitro of polypeptide involved in the present invention can be identified by measuring its minimum inhibitory concentration (MIC).National Committee of Clinical Laboratory Standards (NCCLS) recommend adoption constant broth dilution method is measured the minimum inhibitory concentration (MIC) of each antibacterial peptide, and substratum adopts Mueller-Hinton (MH) broth culture.Using amphotericin B, Polymyxin E and vancomycin as positive control.Active determination in vitro shows, polypeptide provided by the invention all has significant sterilization effect to gram-positive microorganism, Gram-negative bacteria and fungi, separated clinically resistance gram-positive microorganism, resistance Gram-negative bacteria and resistance fungi are had to significant sterilization effect equally, there is wide spectrum, efficient feature.Therefore polypeptide involved in the present invention can be treated as the alternative traditional microbiotic of activeconstituents clinically.
Following examples only represent the aspect that the present invention sets forth, and are not the limitations of theme of the present invention.
Embodiment
The preparation of embodiment 1:AMP-1 and purifying
Sequence: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ ID NO:3)
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
The amino acid of required band protection is Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Phe-OH and Fmoc-L-Tyr (tBu)-OH.
Reagent: HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 type Peptide synthesizer, Waters600 Semipreparative chromatography instrument, magnetic stirring apparatus.
(3) operation steps (take 0.25mmol as example)
A. solid state chemistry synthesizes polypeptide
Take Rink Amide mbha resin 0.61g; be placed in the reactor of Peptide synthesizer; add 10mLDMF; soak 2h; then add 20%PIP/DMF solution 15mL; mix 30min and remove amino protecting agent; with DMF washing resin 7 times; then in reactor, add 387.4mg Fmoc-L-Phe-OH, equimolar coupling reagent DIC (0.33mol/L) to react with HOBt (0.33mol/L); temperature of reaction is room temperature; with ninhydrin reaction monitoring reaction process, guarantee that amino acid is coupled on resin, use DMF washing resin 7 times.After first amino acid is coupled on resin, can proceed according to the method described above next amino acid whose linked reaction, so circulation, until all amino acid coupling completes.
B. cracking and precipitation
After peptide end of synthesis, vacuum-drying resin, weighs.Ratio according to 1g resin 10mL lytic reagent adds lytic reagent, and reagent proportioning is TFA: thioanisole: 1,2-ethandithiol: 75% phenol: water=85: 5: 5: 4: 1, and stirring at room reaction 3 hours, suction filtration.Then to the ice ether sedimentation polypeptide that adds 10 times of volumes in cracking suction filtration liquid, centrifugal, abandon supernatant, then with ice ether repetitive scrubbing, precipitate 4~5 times vacuum-drying, the thick peptide of weighing.
C. reverse-phase chromatography purifying
With preparation HPLC, adopt reverse-phase chromatography, the above-mentioned thick peptide of purifying.
HPLC condition is as follows:
Chromatographic column: XBridgeTM Prep C18 5 μ m OBDTM 19 * 150mm
Flow velocity: 10mL/min
Detect wavelength: 210nm
Moving phase: A: containing the 0.1%TFA aqueous solution
B: containing the acetonitrile of 0.1%TFA
Gradient elution program is as table 3:
Table 3 gradient elution table
Collect the part that target peptide purity is greater than 99%, then 50 ℃ are evaporated to dry.Through ESI-MS, measure, the molecular weight of this peptide is 1880.28, and theoretical value is 1880.43.
Embodiment 2: the preparation of HRP5 analogue and purifying in table 4
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
Amino acid Fmoc-L-Arg (Pbf)-OH, Fmoc-D-Arg (Pbf)-OH, Fmoc-L-Leu-OH, the Fmoc-L-HoR (Pbf) of required band protection-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-D-Lys (Boc)-OH, Fmoc-L-Phe-OH, Fmoc-D-Phe-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Nle-OH, Fmoc-L-Thi-OH and Fmoc-L-Aib-OH.
Reagent: HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 type Peptide synthesizer, Waters600 Semipreparative chromatography instrument, magnetic stirring apparatus.
(3) operation steps (take 0.25mmol as example)
With the method preparation of operation steps a-c in similar embodiment 1 and the polypeptide in purification Table 4, collect the part that purity is greater than 99%, then 50 ℃ are evaporated to dry.ESI-MS measured value is as shown in table 4.
Table 4HRP5 analogue
Embodiment 3: the preparation of cyclic peptide AMP-13 and purifying
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
The amino acid of required band protection is Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Tyr (tBu)-OH and Fmoc-L-Cys (Trt)-OH.
Reagent: HOBt, DIC, DMF, piperidines, 30%H
2o
2.
(2) instrument
PSI300 type Peptide synthesizer, Waters600 Semipreparative chromatography instrument, magnetic stirring apparatus.
(3) operation steps
A. method preparation and the purifying with operation steps a-c in similar embodiment 1 has following sequence (Cys-Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-Cys-NH
2) polypeptide, collect the part that purity is greater than 99%, then 50 ℃ are evaporated to dry.Through ESI-MS, measure, the molecular weight of this peptide is 2086.24, and theoretical value is 2086.72.
B. the formation of intramolecular disulfide bond
Take polypeptide 100.0mg in the present embodiment operation steps a, be dissolved in 200ml water, be mixed with the solution that concentration is 0.5mg/ml, adding 3.33ml concentration is 30% hydrogen peroxide, stirring reaction under room temperature, with the carrying out of HPLC monitoring reaction, until be converted into oxidized form completely.
C. reverse chromatograms purifying
With the method purifying of operation steps c in similar embodiment 1, collect the part that cyclic peptide AMP-13 purity is greater than 99%, then 50 ℃ are evaporated to dry.Through ESI-MS, measure, the molecular weight of this peptide is 2084.25, and theoretical value is 2084.72.
Embodiment 4: antibacterial activity in vitro is measured
The minimum inhibitory concentration (MIC) of each antibacterial peptide of constant broth dilution method determination of recommending according to National Committee of Clinical Laboratory Standards (NCCLS), substratum adopts Mueller-Hinton (MH) broth culture.
Concrete steps are:
(1) antibacterials stock solution preparation:
Accurately compound concentration is above-mentioned HRP5 analogue AMP 1~13 and positive reference substance amphotericin B, Polymyxin E and the vancomycin of 1280 μ g/mL.Each stock solution preparing is placed in-20 ℃ of environment and saves backup.
(2) preparation of substratum:
Take MH broth culture 21g, be dissolved in distilled water and be settled to 1L, 121 ℃ of high-temperature sterilization 30min.
(3) preparation of inoculum:
With 3~5 of transfering loop picking plesiomorphism bacterium colonies to be checked, be inoculated in the MH meat soup of 4~5mL, hatch 2~6h for 35 ℃.Increase logarithmic phase bacterium liquid after bacterium with physiological saline or MH meat soup corrected concentrations to 0.5 Maxwell than turbid standard, approximately containing 1~2 * 10
8cFU/mL.With MH meat soup, above-mentioned bacteria suspension is carried out to dilution in 1: 100 rear standby.
(4) preparation of dilution antibacterials and the inoculation of bacterium liquid:
Get 13 of sterile test tube (13 * 100mm), be arranged in a row, in the 1st pipe, add 1.6mL MH meat soup, in 2-13 pipe, respectively add 1mL MH meat soup, then to the 1st pipe, add antibacterials stoste (1280 μ g/mL) 0.4mL, mix, then draw 1mL to the 2 pipes, after mixing, from the 2nd pipe, draw again 1mL to the 3 pipes, like this continuously doubling dilution to the 11 pipes, and from the 11st pipe, draw 1mL and discard, then in 1-11 pipe and the 13rd pipe, add above-mentioned each 1mL of the inoculum preparing, make the final bacterial concentration of every pipe be about 5 * 10
5cFU/mL.1-11 pipe drug level is respectively 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, the 12nd manages as not containing the blank of antibacterials and inoculum, and the 13rd pipe is not for containing the negative control of antibacterials.
(5) hatch: by the good stopper of dilution pipe close of having inoculated, put in 35 ℃ of normal air incubators and hatch 16~20h.
(6) result: with visual inspection, be the minimum inhibitory concentration (MIC) of this sample without the lowest drug concentration of bacterial growth.The MIC measurement result of each antibacterial peptide is as shown in table 5.
The MIC measurement result of table 5HRP5 analogue