CN106496303A - The peptide for inhibiting of metal beta lactamase and its application - Google Patents

Abstract

The present invention relates to biomedicine field, and in particular to a kind of inhibitor tri-peptides of 1 type metal beta lactamases of NDM, its aminoacid sequence is：Phe‑Cys‑Pro.Pharmacodynamics test shows that the peptide of the present invention can be used for preparation antibacterials to be used for preventing and treating drug-fast bacteria infection.

Description

The peptide for inhibiting of metallo-β-lactamase and its application

Technical field

The present invention relates to a kind of inhibitor tri-peptides of metallo-β-lactamase, more particularly to the peptide prepare antibacterials and Purposes in prevention, treatment drug-fast bacteria infection.

Background technology

It is to cause antibacterial especially one of important mechanisms of gram negative bacteria drug resistance to produce beta-lactamase, and they can water The beta-lactam nucleus of solution beta-lactam antibiotic, cause antibiotic to inactivate.Beta-lactamase can be divided into serine β-interior acyl Amine enzyme and metallo-β-lactamase (MBLs).Dependency according to the homology of aminoacid sequence and to metal Zn ions, MBLs is further divided into B1, B2 and B3 subclass.Have now been found that B1 classes have 18 homologous families, B2 classes to have 3 homologous families, B3 Class has 9 homologous families.As metallo-β-lactamase can be hydrolyzed including nearly all including the dilute class antibiotic of carbon penicillium sp Beta-lactam antibiotic, and list currently without effective inhibitor, therefore MBLs is current clinically anti-infective therapy Significant challenge, receive extensive concern.

IMP, VIM in B1 classes MBLs and NDM types metallo-β-lactamase clinic recall rate highest popular in recent years. In August, 2010《Lancet-infectious disease》Report on magazine and isolate and obtain 180 plants and contain in India, Pakistan and Britain The antibacterial of blaNDM-1 plasmid genes, experiment find, they in addition to tigecycline and polymyxin, to other all antibiosis Element all has height drug resistance.The mutant that constantly evolves causes clinical treatment more difficult, and therefore exploitation wide spectrum MBLs suppresses Agent becomes a major challenge of medical domain.

Content of the invention

The present invention is with the metallo-β-lactamase produced by multidrug resistance antibacterial as target, and is directed to such enzymatic hydrolysis The active center design of antibiotic has synthesized a tripeptides, and the tripeptides of the present invention can combined with antibiotic treatment multidrug resistance antibacterial sense Dye.

The metallo-β-lactamase inhibitor tri-peptides of the present invention are wide spectrum metallo-β-lactamase peptide for inhibiting, it is characterised in that Length is 3 aminoacid, and topological structure is linear, and chemical structural formula is：

Metallo-β-lactamase inhibitor tri-peptides involved in the present invention are prepared with solid-phase synthesis, and method is simple, technology Maturation, product quality are easily controlled, and can meet the needs of large-scale industrial production.

Pharmacodynamics test proves the tripeptides of the present invention to typical metallo-β-lactamase, such as NDM-1, IMP-1 and VIM-2 Deng with obvious inhibitory action.

Pharmacodynamics test proves that the tripeptides of the present invention and beta-lactam antibiotic have good synergism, can be substantially Fastbacteria is suppressed to produce and be greatly lowered the minimal inhibitory concentration (MIC) of beta-lactam antibiotic.Described β-interior acyl The preferred Cefuroxime Sodium of amine antibiotic.

The tripeptides of the present invention can also be combined with pharmaceutically acceptable salt, and equally there is its drug effect.These salt include (but It is not limited to) salt that formed with alkali metal or alkaline-earth metal (such as sodium, potassium, calcium or magnesium).Other salt include and following mineral acid shape Into salt：Such as hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, and the salt formed with organic acid, and organic acid then refers to acetic acid, oxalic acid, fourth two Acid, tartaric acid, methanesulfonic acid and maleic acid.Pharmaceutically acceptable salt particular certain cancers.

The invention discloses a kind of antibacterial combination, wherein containing the tripeptides or its pharmaceutically acceptable salt and medicine Acceptable carrier on.

Aforementioned pharmaceutical compositions can make injection, tablet, injectable sterile powder, powder, granule, capsule, Multiple dosage forms such as oral liquid, unguentum, cream.Above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.Can be with Muscle, endothelium, subcutaneous, vein, mucosal tissue are imported by injection, oral, collunarium, eye drip, the method for physically or chemically mediating, Or human body is imported by other material mixings or after wrapping up.

Usually, tripeptides using dosage of the invention is at 0.001～10 gram/day, it is also possible to deviateed according to the situation of disease This dosage range.

Part pharmacodynamics test and the result of the tripeptides of the present invention is presented herein below：

First, tripeptides is to NDM-1 type metallo-β-lactamase inhibitory activity (IC₅₀) measure：

With Cefuroxime Sodium as reporter substrate, NDM-1 types metallo-β-lactamase hydrolysis substrate is determined under 274nm wavelength The change of absorbance afterwards.Measure buffer be 50mM HEPES, 250mM NaCl, 2 μM of ZnCl₂, pH=7.25,30 DEG C of temperature.

(1) metallo-β-lactamase (10 μM of final concentration) is incubated 5min first in buffer so that Zn²⁺Fully occupy Active center；

(2) inhibitor tri-peptides of 10 μ L variable concentrations, 30 DEG C of incubation 5min are added inhibitor is fully combined with enzyme；

(3) system is proceeded in quartz colorimetric utensil, is surveyed while adding 8 μ L Cefuroxime Sodium (160 μM of final concentration) immediately Determine the change of system absorbance, record data.

As substrate Cefuroxime Sodium is declined by its absorbance after enzyme hydrolysiss, thus can be with by the change of mensuration absorbance Characterize the hydrolysis degree of substrate.The inhibitor tri-peptides for calculating variable concentrations hydrolyze the suppression ratio of substrate to NDM-1, are suppressed with tripeptides The concentration of agent is calculated IC of the tri-peptide molecule of the present invention to NDM-1 type metalloenzyme to suppression ratio matched curve₅0 value is 86.46 μM, show which has good inhibiting effect to NDM-1 type metalloenzyme.

2nd, inhibitor tri-peptides are to IMP-1 type metallo-β-lactamase inhibitory activity (IC₅₀) measure：

With Cefuroxime Sodium as reporter substrate, IMP-1 types metallo-β-lactamase hydrolysis substrate is determined under 274nm wavelength The change of absorbance afterwards.Measure buffer be 50mM HEPES, 250mM NaCl, 2 μM of ZnCl₂, pH=7.25,30 DEG C of temperature. Concrete operation step is with embodiment 1.The inhibitor tri-peptides for calculating variable concentrations hydrolyze the suppression ratio of substrate to IMP-1, with tripeptides The concentration of inhibitor is calculated IC of the tri-peptide molecule to IMP-1 type metalloenzyme to suppression ratio matched curve₅₀49.6 μM of value, says This tri-peptide molecule bright has good inhibiting effect to IMP-1 type metalloenzyme.

3rd, tri-peptide molecule and beta-lactam antibiotic cooperate with the measure of anti-NDM-1 types engineering bacteria activity：

Synergistic antimicrobial experiment is carried out in order to safety, using comparatively safe genetically engineered E.coli BL21 (DE3)/pET28a-NDM-1 bacterial strains as Resistant strain, using E.coli BL21 (DE3)/pET28a bacterial strains as negative control. Minimal inhibitory concentration (Minimum Inhibitory Concentration, MIC) is determined using doubling dilution, by MIC The change reflection peptide for inhibiting of value and the synergetic antibacterial effect of antibiotic.

Concrete grammar is as follows：

By engineering bacterial strain E.coli BL21 (the DE3)/pET28a-NDM-1 preserved in ultra cold storage freezer and control bacterium Strain E.coli BL21 (DE3)/pET28a is inoculated in Luria-Bertani (LB) solid medium of sterilizing respectively, in 37 DEG C Culture 12 hours is inverted in constant incubator.It is transferred in the LB liquid medium of sterilizing respectively with inoculating loop picking single bacterium colony, In 37 DEG C, under the conditions of 200rpm, concussion and cultivate is to exponential phase.Bacterium solution at 600nm wavelength is determined with ultraviolet spectrophotometer Absorbance (OD₆₀₀), according to 1OD=1 × 10⁹The conversion relation of CFU/mL, respectively by the engineering of expression metallo-β-lactamase Bacteria strain and control strain are diluted to final concentration 1 × 10⁶CFU/mL.

Matched group (1)：E.coli BL21 (DE3)/pET28a bacterium.Doubling dilution successively is added in aseptic 96 orifice plate The antibiotic Cefuroxime Sodium of final concentration of 512～2 μ g/mL, then adds final concentration 1 × 10 in each hole⁶The bacterium of CFU/mL Liquid, mix homogeneously.

Matched group (2)：Engineering bacteria E.coli BL21 (the DE3)/pET28a-NDM-1 of expression metallo-β-lactamase.? The antibiotic Cefuroxime Sodium of final concentration of 512～2 μ g/mL of doubling dilution successively is added in aseptic 96 orifice plate, then to each Final concentration 1 × 10 is added in hole⁶The bacterium solution of CFU/mL, mix homogeneously.

Matched group (3)：Engineering bacteria E.coli BL21 (the DE3)/pET28a-NDM-1 of expression metallo-β-lactamase.? Final concentration 1 × 10 is added in aseptic 96 orifice plate⁶The inhibitor tri-peptides of the bacterium solution of CFU/mL and final concentration of 200 μM, mix homogeneously.

Experimental group is using engineering bacteria E.coli BL21 (the DE3)/pET28a-NDM- that can express metallo-β-lactamase 1.

Experimental group (1)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The inhibitor tri-peptides of the bacterium solution of CFU/mL and final concentration of 200 μM, Mix homogeneously.

Experimental group (2)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The inhibitor tri-peptides of the bacterium solution of CFU/mL and final concentration of 100 μM, Mix homogeneously.

Experimental group (3)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The inhibitor tri-peptides of the bacterium solution of CFU/mL and final concentration of 50 μM, Mix homogeneously.

Experimental group (4)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The inhibitor tri-peptides of the bacterium solution of CFU/mL and final concentration of 25 μM, Mix homogeneously.

Experimental group (5)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The tripeptides of the bacterium solution of CFU/mL and final concentration of 12.5 μM suppresses Agent, mix homogeneously.

Experimental group (6)：The concentration for adding doubling dilution successively in aseptic 96 orifice plate is the antibiotic head of 512～2 μ g/mL Spore cefuroxime sodium, then adds final concentration 1 × 10 in each hole⁶The tripeptides of the bacterium solution of CFU/mL and final concentration of 6.25 μM suppresses Agent, mix homogeneously.

In 37 DEG C of slow concussion and cultivates 16 hours, it was the absorbance at 600nm to determine wavelength to above each group.Can't detect The final concentration of minimal inhibitory concentration (MIC) of Cefuroxime Sodium in the hole of bacterial growth, tri-peptide molecule Phe-Cys-Pro is to containing The Resistant strain Combination effect of NDM-1 is as shown in table 1.

1 tripeptides Phe-Cys-Pro of table cooperates with the measurement result of anti-NDM-1 engineering bacterias with Cefuroxime Sodium

^a-：Tripeptides Phe-Cys-Pro itself is without antibacterial activity.

As seen from the results in Table 1, in matched group (1), Cefuroxime Sodium has well to sensitive strain E.coli BL21 (DE3) Inhibitory action (2 μ g/mL of MIC ＜)；Matched group (2) be Resistant strain E.coli BL21 (DE3)/pET28a-NDM-1, cephalo Cefuroxime sodium can hardly suppress the growth (MIC=256 μ g/mL) of the bacterial strain, while also demonstrating the bacterial strain for fastbacteria；Control In group (3) containing final concentration of 200 μM Phe-Cys-Pro and do not contain antibiotic, culture 16 hours after bacterium solution very muddy, card Bright inhibitor tri-peptides itself are without antibacterial action.

Can be seen that from the MIC of experimental group (1)～(6) and be used in combination with antibiotic Cefuroxime Sodium as Phe-Cys-Pro When, the antibacterial activity that Cefuroxime Sodium can be significantly improved by synergism, when inhibitor tri-peptides concentration is 200 μM, cephalo The antibacterial activity (MIC value) of cefuroxime sodium improves 16 times (256/16)；When inhibitor tri-peptides concentration is 25 μM, Cefuroxime Sodium Antibacterial activity (MIC value) improve 2 times (256/128).

Specific embodiment

Embodiment 1

The preparation of Phe-Cys-Pro

1. 2.0g MBHA (4- toluene hydrogen amine) resin is weighed in the reaction tube of clean dried, add appropriate DMF (diformazans Base Methanamide), then activation 30min or so weighs the Phe 1mmol that Fmoc (fluorenes methoxy carbonyl acyl group) is protected, DMAP (4 diformazans Aminopyridine) 150mg, the DIC for plus 98% (N, N '-DIC) in reaction tube, DMF does solvent reaction to 1ml 3h.Reaction is finished is washed 4～6 times with DMF, adds appropriate pyridine and acetic anhydride, and volume ratio is 1: 1, reacts 30min.Reaction is finished 4～6 times are washed with DMF.Then the piperidine solution with 20% takes off the Fmoc of aminoacid, takes off common twice 15min, 10min+5min. Washed 4 times with DMF again, methanol is washed 2 times, take out a small amount of resin 1,2,3-indantrione monohydrate detectable and detect, be detected as blueness, you can carry out down Single step reaction.

2. weigh Fmoc protection Cys 3mmol, HBTU (BTA N, N, N ', N '-tetramethylurea hexafluorophosphoric acid Ester) in reaction tube, addition DIEA (DIPEA) 0.5mL reacts 40min, is washed 4～6 times with DMF, taken 3mmol A small amount of resin detects that with 1,2,3-indantrione monohydrate detectable show colourless, the piperidine solution for being subsequently adding 20% takes off Fmoc, 10min+5min, Then washed 4 times with DMF, methanol is washed twice, take out a small amount of resin 1,2,3-indantrione monohydrate detectable and detect, be detected as blueness, you can enter Row next step is reacted.

3. the C-terminal aminoacid Pro 3mmol of Fmoc protections are weighed, and HBTU 3mmol add DIEA in reaction tube 0.5mL, reacts 40min, is washed 4～6 times with DMF, takes a small amount of resin 1,2,3-indantrione monohydrate detectable detection, shows colourless, be subsequently adding 20% piperidine solution de- Fmoc, 10min+5min, are then washed 4 times with DMF, and methanol is washed twice, takes out a small amount of resin indenes three The detection of ketone detectable, is detected as blueness.

4. last use trifluoroacetic acid cutting liquid cuts 2h, and reactant liquor sucking filtration obtains the trifluoroacetic acid solution of polypeptide, heavy with ether Form sediment, then centrifugation is washed 3～5 times with ether again, obtain white solid, through C18 reverse phase preparative column desalinations, lyophilizing, taken Analysis.HPLC determines the purity of tripeptides and identifies that its molecular weight is 365.10 for 95.56%, MS, with 365.45 base of theoretical molecular This is consistent.

Embodiment 2

The preparation (capsule) of the antibacterial combination containing Phe-Cys-Pro peptide for inhibiting

According to the consumption of 100 capsules, weigh above each adjuvant respectively finely ground sieve after mix homogeneously, then passed with equivalent Addition adds Cefuroxime Sodium and Phe-Cys-Pro, is fully ground so as to be uniformly dispersed, after 80 mesh sieves, then records plastic Capsule.

Claims (5)

1. the peptide or its pharmaceutically acceptable salt of following three aminoacid sequences are contained：Phe-Cys-Pro.

2. a kind of antibacterial combination, peptide or its pharmaceutically acceptable salt wherein containing claim 1 and pharmaceutically may be used The carrier of acceptance.

3. a kind of antibacterial combination, wherein containing beta-lactam antibiotic, the peptide of claim 1 or its can pharmaceutically connect The salt that receives and pharmaceutically acceptable carrier.

4. the peptide of claim 1 or its pharmaceutically acceptable salt are used for the purposes for preparing antimicrobial drug.

5. the purposes of claim 4, wherein antibacterials are the medicines for preventing or treating drug-fast bacteria infection disease.