Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • p2y inhibitor Introduction Antimicrobial peptides AMPs are a

    2018-11-01

    Introduction Antimicrobial peptides (AMPs) are a group of biomolecules that have evolved to recognize and kill target microbes by binding to and disrupting cell membranes. Several unique characteristics of AMPs make them attractive alternatives to antibodies for detection of microbial biothreats: resistance to proteases; stability to environmental extremes; and high affinity, overlapping (but not identical) binding interactions with microbial membranes and membrane components. Arrays of AMPs have been used to detect and classify microbial pathogens with similar sensitivity to antibody-based assays; their broad-spectrum binding activities also provide the potential for detection of unknown microbes [11,12,16,18,27]. In previous studies, surface-immobilized AMPs mediated target binding, and an additional “tracer” (e.g., labeled antibody or non-specific dye) was required for signal transduction. This constraint increases the number of reagents required and the overall complexity of the assay. Development of an AMP-based material that is capable of both target recognition and signal generation without addition reagents or processing steps is highly desirable. This type of construct would provide greatly enhanced potential for application of AMP-based detection techniques in autonomous and distributed sensing platforms. A number of publications report use of porphyrin-peptide conjugates for targeting and photodestruction of cells [3–5,7,10,13,24]. In these studies, the antimicrobial peptide domain is used to interact with the appropriate cell (cancer cell, Gram-negative bacterial pathogen), while the porphyrin moiety is used as a source of reactive oxygen species upon illumination [21]. Porphyrins are large macrocyclic compounds with strong absorbance and fluorescence characteristics. They have been applied in a wide variety of detection approaches due to the sensitivity of those characteristics to their immediate environment. Spectrophotometric and binding characteristics can be altered through modification of the porphyrin structure. Several reports have described modifications using single p2y inhibitor or dipeptides [2,29,33]. Binding of proteins by these porphyrin derivatives resulted in changes to their fluorescence characteristics, and arrays of the constructs were applied to discrimination of proteins. Modification of the periphery of a porphyrin using cytosine was similarly applied to detection of guanine [6]. Other works have shown that porphyrins can be used to report conformational changes in enzymes upon substrate binding when the porphyrin-enzyme interaction results in competitive or mixed-type enzymatic inhibition [30,31]. This study sought to demonstrate the potential for antimicrobial peptides modified using porphyrins in indication of the presence of bacterial cells. The goal was development of constructs providing an avenue for achieving reagentless detection and classification of bacterial targets. Sensing in this case would utilize changes in the local environment of a covalently attached porphyrin resulting from conformational changes in the antimicrobial peptide. While others have proposed application of porphyrin-peptide conjugates as imaging agents (e.g., [13]), this approach would provide the potential for use of an array of peptide-porphyrin conjugates in detection of bacteria with broad classification of the detected cells based on the differential changes in the spectrophotometric characteristics of the porphyrin-peptide conjugates. Here, synthesis and characterization of a set of four porphyrin-AMP constructs is presented. Their utility with regard to the potential for indication of bacterial targets is discussed.
    Methods 5-Mono(4-carboxyphenyl)-10, 15, 20-triphenyl porphine (C1TPP) was obtained from Frontier Scientific (Logan, UT). Vanadium (III) bromide, zinc chloride, cobalt (II) chloride, and dimethylsufloxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimidyl ester (NHS) and sulfo-NHS were purchased from Pierce Thermo Scientific (Rockland, IL). Antimicrobial peptides indolicidin (Ind), bactenecin (Bac), and cecropin A (1–8)-melittin (1–18) hybrid peptide (CeMe) were purchased from American Peptide Company (Sunnyvale, CA); polymyxin E (PME) was obtained from Sigma-Aldrich. Sequences are provided in Table 1.