KPC (Klebsiella pneumoniae Carbapenemase) inhibition peptide and application thereof

A carbapenemase and inhibitory peptide technology, which is applied in the field of KPC carbapenemase inhibitory peptides, can solve the problem that the inhibitory effect of β-lactamase is not obvious, and achieve good KPC carbapenemase inhibitory activity, The effect of low production cost and good development prospects

Inactive Publication Date: 2016-01-20
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The currently clinically used β-lactamase inhibitors (clavulanic acid, sulbactam, tazobactam) have less and less obvious inhibitory effects on emerging new β-lact...

Method used

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  • KPC (Klebsiella pneumoniae Carbapenemase) inhibition peptide and application thereof
  • KPC (Klebsiella pneumoniae Carbapenemase) inhibition peptide and application thereof
  • KPC (Klebsiella pneumoniae Carbapenemase) inhibition peptide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The preparation of embodiment 1KPC carbapenemase inhibitory peptide

[0033] The amino acid sequence of the KPC carbapenemase inhibitory peptide is Pro-Asn-Gln-Trp, and the chemical structure is as follows figure 1 shown.

[0034] KPC carbapenemase inhibitory peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl (FMOC) as the N-terminal protecting group. Cleavage it from MBHA resin with a mixture of 92% trifluoroacetic acid, 5% water and 3% triisopropylsilane (TIA) (92%, 5%, and 3% are all mass percentages). After multiple precipitations of diethyl ether, it was purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Use C18 reverse-phase preparative column (20mm * 250mm, 5 μm); Mobile phase: 0.06% trifluoroacetic acid, 0%-80% (0.06%, 0%-80% are volume percentage) acetonitrile is mobile phase, 1.5mL / min Gradient elution was performed at a flow rate. RP-HPLC detection showed that its purity was >98%, and it w...

Embodiment 2

[0035] Example 2 KPC Carbapenemase Inhibiting Peptide Determination of KPC-2 Carbapenemase Inhibitory Activity

[0036] With meropenem as the reporting substrate, the experiment was divided into 3 groups, and the temperature was kept at 37±1°C:

[0037] (1) Meropenem + KPC-2 carbapenemase group: Add 5 μL of 2 mmol / L meropenem and 90 μL of 30 mmol / L HEPES buffer (pH 7.4) to a mixed solution of 5 μL 1 μmol / L of KPC-2 carbapenemase in a total volume of 100 μL.

[0038] (2) Meropenem + KPC-2 carbapenemase + enzyme inhibitory peptide group: add 5 μL of 2 mmol / L meropenem and 80 μL of 30 mmol / L HEPES buffer (pH 7.4) mixed solution Add 5 μL of KPC-2 carbapenemase at a concentration of 1 μmol / L and 10 μL of KPC carbapenemase inhibitory peptide at a concentration of 1 μmol / L for a total volume of 100 μL.

[0039] (3) Meropenem group: 95 μL of 30 mmol / L HEPES buffer (pH 7.4) was added to 5 μL of 2 mmol / L meropenem, with a total volume of 100 μL.

[0040] Since the absorbance of the r...

Embodiment 3

[0041] Example 3 KPC Carbapenemase Inhibiting Peptide and β-lactam Antibiotic Synergistic Antibacterial Action Determination

[0042] In order to carry out synergistic antibacterial experiments safely, the relatively safe genetically engineered strain E.coliRosetta(DE3) / pET28a-KPC-2 was used as a drug-resistant strain, and the E.coliRosetta(DE3) strain was used as a negative control. The minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) is determined by the double dilution method, and the synergistic antibacterial effect of the KPC carbapenemase inhibitory peptide of the present invention and β-lactam antibiotics is reflected by the change of the MIC value.

[0043] The specific method is as follows:

[0044] The engineered bacterial strain E.coliRosetta (DE3) / pET28a-KPC-2 (drug-resistant engineered bacteria) and the control strain E.coliRosetta (DE3) preserved in an ultra-low temperature refrigerator at -80°C can express KPC-2 carbapenemase They were r...

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Abstract

The invention discloses a KPC (Klebsiella pneumoniae Carbapenemase) inhibition peptide and an application thereof and belongs to the field of biological medicines. The amino acid sequence of the KPC inhibition peptide is Pro-Asn-Gln-Trp, and the KPC inhibition peptide can be prepared with a solid-phase synthesis method and has a remarkable inhibiting effect on KPC. Through combined utilization of the inhibition peptide and beta-lactam antibiotics, the antibiotics can be prevented from being hydrolyzed and damaged by the KPC and losing efficacy, and the inhibition peptide can be used for preparing drugs for preventing and/or treating diseases caused by drug-resistant bacteria infection and/or antibacterial agents. The KPC inhibition peptide has higher enzyme inhibitory activity and low hemolytic activity, realizes artificial synthesis conveniently, is suitable for industrialization, provides a new choice for development of new antibacterial agents and compound preparations and has the good development prospect in the field of treatment of the drug-resistant bacteria infection.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a KPC carbapenemase inhibitory peptide and its application. Background technique [0002] In 2013, the US Centers for Disease Control and Prevention announced the priority development of antibacterial drugs for the treatment of carbapenem-resistant Enterobacteriaceae (Carbapenem-resistant Enterobacteriaceae, CRE). The reason is that the emergence and spread of CRE bacteria producing KPC carbapenemase (Klebsiellapneumoniae Carbapenemase, KPC) seriously reduces the clinical efficacy of β-lactam antibiotics (including carbapenem antibiotics), making clinical treatment Drug-resistant bacterial infections are facing severe challenges. CREs are often resistant to most antibiotics, and the headache for medical staff is that this type of drug-resistant bacteria is sometimes only sensitive to the more toxic antibacterial agent colistin. [0003] Among CREs, the Klebsiella genus (Klebsiellaspp...

Claims

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Application Information

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IPC IPC(8): C07K5/117C07K1/04A61K38/07A61P31/04
Inventor 沈秉正高翔祝成亮宋金春曾智彭燕
Owner WUHAN UNIV
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