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Use of bacterial beta-lactamase for in vitro diagnostics and in vivo imaging, diagnostics and therapeutics

A lactamase, bacterial technology with applications in pathogenic microbiology and imaging, pharmaceutical fields

Inactive Publication Date: 2013-04-10
TEXAS A&M UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More specifically, the prior art is deficient in sensitive and specific real-time optical imaging methods for β-lactamase-positive bacteria that can be used both in vitro and in living subjects to Diagnose and localize bacterial infections, rapidly screen for new therapies, and identify new drug targets

Method used

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  • Use of bacterial beta-lactamase for in vitro diagnostics and in vivo imaging, diagnostics and therapeutics
  • Use of bacterial beta-lactamase for in vitro diagnostics and in vivo imaging, diagnostics and therapeutics
  • Use of bacterial beta-lactamase for in vitro diagnostics and in vivo imaging, diagnostics and therapeutics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Bla detection in Mycobacterium tuberculosis in culture

[0105] Using whole cells grown to early log phase and whole cell lysates (lysates), for the detection of Bla in Mycobacterium tuberculosis, compare potential fluorescent gene substrate compounds to known compounds, including nitrocefaxime (Calbiochem), CENTA Bla substrate (Calbiochem), Fluorocillin Green (Molecular Probes), CCF2-AM (Invitrogen) and CCF4-AM (Invitrogen). Dilutions were analyzed for all of these samples to determine the lowest number of bacteria or amount of lysate that produced a significant signal. Potency assays were performed before and after assays with whole cells and prior to lysis for lysates to determine the number of CFUs actually used. Sensitivity and reproducibility were assessed spectrophotometrically in quadruplicate using 96-well plates incubated in bacterial culture medium at 37°C for 15-120 minutes. Initially, compounds were used at the concentrations suggested by the manufactur...

Embodiment 2

[0137] Intra-vital microscopy imaging using cell transplantation models

[0138] Generic donor Tr, CD8+ T cells, monocytes, macrophages, and dendritic cells were transplanted into syngeneic mice infected with BCG, and bioluminescence imaging (BLI) in vivo and image-guided biopsy The temporal distribution of these cells was imaged by endoscopy (IVM). A transgenic mouse line in which luciferase is produced through the β-actin promoter provides a source of tissues and cells that will glow in non-transgenic animals (11-12). This mouse line (L2G85), exhibits bright bioluminescence from firefly luciferase (Fluc), and weak GFP fluorescence, so it is in contrast to a single cell that exhibits strong GFP expression and fluorescence in lymphocytes. Germline mating. Thus, after the spatial distribution of the universal donor stem cells and other cells, their exhibited BLI, redistribution or will be cleared in the recipient can be performed and the detected cells can be subsequently o...

Embodiment 3

[0148] Crystallization of enzymes from Mycobacterium tuberculosis BlaC and BlaC mutants

[0149] Very good BlaC crystals were obtained after several months of crystallization. Use 0.1M Tris-HCl, pH8.0, 20.MNH 4 h 2 PO 4 crystallization conditions to prepare co-crystals with penicillin. These crystals allow the visualization of active sites and intermediates of the intact protein, but the initial bound substrates are not visible due to the turnover of the crystal itself. To overcome this obstacle, the Mtb BlaC mutant enzyme was constructed with a mutation in the Glu residue (E166A) involved in hydrolysis, which allowed visualization of specific interactions required for trapping and catalysis of the acy-intermediate on the enzyme. This mutant has been crystallized using a rapid (i.e., about two weeks) crystallization process that yields a high-quality Mtb BlaC mutant ready to be taken up by a substrate ( Figure 1A ). This demonstrates that substrate incorporation into M...

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Abstract

Provided herein are methods for detecting, quantifying, differentiating, diagnosing and imaging pathogenic bacteria or condition associated therewith using substrates for bacterial enzymes. Fluorescent, luminescent or colorimetric signals emitted by substrates or enzyme products in the presence of the bacteria are compared to controls to detect and locate the pathogenic bacteria. Provided is a method for screening therapeutic agents to treat the pathophysiological conditions by measuring a signal emitted from the substrates or products in the presence and absence of the potential therapeutic agent and a diagnostic method for detecting a mycobacterial infection in a subject by contacting biological samples with a substrate and imaging for signals emitted from a mycobacterial beta-lactamase product.; Also provided are fluorogenic substrates or substrates comprising a colored dye or a chemical reagent effective to induce a color or pH change.

Description

[0001] Cross References to Related Applications [0002] This international application claims priority under 35 U.S.C. §120 to the pending continuation-in-part patent application of U.S. Serial No. 12 / 802,340, filed June 4, 2010, pursuant to 35 U.S.C. §120 claims priority to pending nonprovisional application U.S. Serial No. 12 / 462,644 filed August 6, 2009, which is claimed pursuant to 35 U.S.C. §119(e) in Priority of U.S. Provisional Application Serial No. 61 / 203,605 filed December 24, 2008 (now abandoned) and U.S. Provisional Application Serial No. 61 / 188,112 filed August 6, 2008 (now abandoned), The entire content of the above application is incorporated by reference herein in its entirety. technical field [0003] The invention generally relates to the fields of medicine, pathogenic microbiology and imaging technology. More specifically, the present invention relates to compounds and reporters for the detection and localization of bacterial pathogens during in vitro or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34C12Q1/04G01N33/52G01N33/15C12R1/32
CPCA61K49/0013A61K49/0021A61K49/0028A61K49/0032A61K49/0041A61K49/0052C12Q1/04C12Q1/34G01N2333/986A61P43/00G01N33/52G01N33/15
Inventor J·D·奇里洛J·C·萨凯帝尼饶江洪谢贺新
Owner TEXAS A&M UNIVERSITY
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