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Identification of lysins and derivatives thereof with bacterial activity against pseudomonas aeruginosa

A lysin, active technology, used in antibacterial drugs, bacterial antigen components, biochemical equipment and methods, etc., can solve problems such as lack of activity

Pending Publication Date: 2020-11-20
CONTRAFECT CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Physiological salts and divalent cations are thought to compete for the LPS binding site and interfere with the α-helical translocation domain of lysins, including GN lysins, thereby limiting activity in blood, and more specifically in the presence of serum , thereby limiting the possibility of using lysins to treat invasive infections (23)
A similar lack of activity in blood has been reported for various OM-permeable and destabilized AMPs (18-20,22)

Method used

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  • Identification of lysins and derivatives thereof with bacterial activity against pseudomonas aeruginosa
  • Identification of lysins and derivatives thereof with bacterial activity against pseudomonas aeruginosa
  • Identification of lysins and derivatives thereof with bacterial activity against pseudomonas aeruginosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1. Bacterial Strains and Growth Conditions. Pseudomonas aeruginosa from human blood obtained from the Hospital for Special Surgery in New York (provided by Dr. Lars Westblade, Professor of Pathology and Laboratory Medicine) was used. Antibacterial screening was performed on a clinical isolate of Monocystis bacterium (CFS-1292). Strain CFS-1292 was cultured in lysogenic broth (LB; Sigma-Aldrich), casamino acid (CAA) medium (5 g / L casamino acid, Ameresco / VWR; 5.2 mM K 2 HPO 4 , Sigma-Aldrich; 1 mM MgSO 4 , Sigma-Aldrich) or CAA supplemented with 25% human serum (AB type, male, pooled; Sigma-Aldrich). For the purposes of this disclosure, the particular isolate of P. aeruginosa is not important and commercially available isolates may be used in this experiment.

Embodiment 2

[0125] Example 2. Gene synthesis and cloning. All lysins and modified lysins were synthesized as gBlocks (IDTTechnologies) and cloned into the arabinose-inducible expression vector pBAD24 by overlap extension PCR or by ligation compatible cohesive ends (24) middle. All constructs were transformed into E. coli strain TOP10 (Thermo Fisher Scientific). Other commercially available expression vectors and systems can be used.

Embodiment 3

[0126]Example 3. Identification of lysins with intrinsic activity. A set of up to 250 putative lysins and lysin-like enzymes were identified in the GenBank database of Pseudomonas aeruginosa genome sequences. 3 search methods were used: i) a targeted BLASTp screen of all Pseudomonas aeruginosa genomes using query sequences of known lysins, ii) a keyword-based search of all annotated Pseudomonas aeruginosa genomes A search, focusing on all superfamily names associated with lysin (and cell wall hydrolases) catalytic and binding domains; and iii) a visual search for lysin-like genes in phage sequences of non-annotated genomes. Once identified, lysin sequences were synthesized as gBlocks, cloned into pBAD24 and transformed into E. coli TOP10 cells. E. coli clones were then examined in a primary antibacterial activity screen (against live Pseudomonas aeruginosa) using an agar-covered plate-based method (11,13) with modifications to allow the assay to include suspension in 50 mM Tri...

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PUM

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Abstract

Disclosed are novel lysin polypeptides active against Gram-negative bacteria, particularly P. aeruginosa, pharmaceutical compositions containing them and methods for their use to treat Gram-negative bacterial infections and more generally to inhibit the growth, or reduce the population, or kill Gram-negative bacteria, including without limitation disrupting biofilms formed by such bacteria. Certain of the disclosed lysins have been modified in amino acid sequence compared to that of lysins by replacement of certain charged amino acids with noncharged amino acids and / or by fusion at the N- or C-terminus with antibacterial peptide sequences with or without an intervening linker.

Description

[0001] Background of the invention [0002] Gram-negative bacteria, especially Pseudomonas ( Pseudomonas ), an important cause of serious and potentially life-threatening invasive infections. Pseudomonas infections constitute a major problem in burns, chronic wounds, chronic obstructive pulmonary disease (COPD) and other structural lung diseases, cystic fibrosis, surface growths on implanted biomaterials, and within hospital surfaces and water supplies, among which It poses many threats to vulnerable patients such as immunosuppressed patients and intensive care (ICU) patients. [0003] Once established in a patient, Pseudomonas aeruginosa ( P. aeruginosa ) can be especially difficult to treat. The genome encodes a number of resistance genes, including multidrug efflux pumps and enzymes that confer resistance to β-lactam and aminoglycoside antibiotics, enabling treatment against this Gram-negative pathogen due to the lack of new antimicrobial therapeutics especially challeng...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K39/02
CPCA61K38/48A61K45/06C12N9/2462A61K9/107A61K9/0075A61K9/0073Y02A50/30A61K38/162C12Y302/01017A61K38/164A61K2300/00A61P31/04A61K38/47
Inventor R·舒赫C·因迪亚尼
Owner CONTRAFECT CORP
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