Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation

Poras, Hervé; Ouimet, Tanja; Orng, Sou-Vinh; Dangé, Emilie; Fournié‐Zaluski, Marie‐Claude; Roques, Bernárd P.

doi:10.1016/j.ab.2011.08.016

Cited by 5 publications

(7 citation statements)

References 47 publications

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“…In this manner, the sets of data that may be useful for design and synthesis of selective inhibitors are obtained (Drąg et al 2010;Kasperkiewicz et al 2012;Poras et al 2011Poras et al , 2013Węglarz-Tomczak et al 2013;Poręba et al 2014).…”

Section: Introductionmentioning

confidence: 99%

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

2015

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Inhibitory potencies of 24 a-aminophosphonic acids against barley seeds (Hordeum vulgare L.) metalloaminopeptidase have been determined to evaluate structural requirements of this enzyme. The enzyme was sensitive mostly to the influence of phosphonic acid analogues of phenylalanine and its homologues, thus showing narrow specificity if compared with porcine aminopeptidases M1 and M17 and with Plasmodium aminopeptidase M17.

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Section: Introductionmentioning

confidence: 99%

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

2015

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“…Synthesis-A quenched fluorescent peptide library corresponding to the general formula Ac-SKG-Pya-(X) n -Nop-GGK-NH 2 has been developed for the characterization of all types of proteolytic activities (15,16). In this formula, Pya (L-pyrenylalanyl) is the fluorescent moiety; Nop (L-4-NO 2 -phenylalanyl) the quencher moiety; X indicates either a single natural amino acid chosen from Ala, Leu, Ile, Glu, Thr, Gln, Phe, Trp, Pro, or Lys or a mixture of these 10 amino acids, and n varies from 0 to 3.…”

Section: Methodsmentioning

confidence: 99%

“…The secreted Msp protease was purified from culture media of L. pneumophila Philadelphia 1 (CIP 103854T-ATCC 33152). The Fluofast library of intramolecularly quenched fluorescent substrates containing the Pya/Nop fluorophore/quencher pair (14,15) was screened in parallel with purified Msp and pseudolysin. Results of this screening provided a lead selective substrate.…”

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confidence: 99%

Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis

Poras

Duquesnoy

Dangé

et al. 2012

Journal of Biological Chemistry

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Background: Legionella pneumophila secretes a protease without known specific substrate and three-dimensional structure.Results: Analysis of a quenched peptide library identified a lead substrate, ameliorated by rational design, using an Msp structural model obtained by the x-ray structure of pseudolysin. Conclusion:The study identifies the first selective substrate for Msp. Significance: This substrate could be useful for Legionella detection.

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“…The fluorigenic substrates of NEP, ACE, and ECE were synthesized using peptide Fmoc chemistry, as previously described. 19,20 The EDTA and phosphoramidon metalloprotease inhibitors were purchased from Sigma.…”

Section: Introductionmentioning

confidence: 99%

“…The culprit region (CP), the stenosic plaque lesion for which the patient had been operated, at the origin of the internal carotid artery, was dissected and separated from the adjacent plaque (stable or noncomplicated plaque, NP), present in the common and external carotid arteries, as previously described. 19,20 Excised segments were immediately rinsed in ice-cold saline. Histological analysis of the samples showed that CPs corresponded to advanced type V or VI atherosclerotic lesions (mainly intraplaque hemorrhage evolving in the necrotic core) while the adjacent area (NP) was characterized as type III or IV lesions (lipid core encapsulated between a fibrous cap and the media) 21−23 according to the classification of Stary et al 24 Samples were rinsed in RPMI 1640, weighed, cut into 2−5 mm 3 pieces, and incubated in culture medium (RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin, and 1% amphotericin) at 37 °C (5% CO 2 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular and Cellular Targets of the MRI Contrast Agent P947 for Atherosclerosis Imaging

et al. 2012

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P947 (DOTA-Gd-peptide) was recently identified as an MRI contrast agent for the detection and characterization of the matrix metalloproteinases (MMP)-rich atherosclerotic plaques. Because this product displays a broad spectrum affinity for the MMP family, we hypothesized that it may also recognize other metalloproteinases overactivated in vulnerable atherosclerotic plaques. Therefore, this study aimed at describing, at the molecular and cellular level, the interactions between P947 and proteases of atherosclerotic plaques. Fluorimetric assays were used to measure the in vitro affinity of P947 toward recombinant and purified MMPs, angiotensin-converting enzyme (ACE), endothelin-converting enzyme (ECE-1), neutral endopeptidase (NEP), and both aminopeptidases A and N (APA and APN). Using similar fluorimetric assays associated with specific substrates, enzymatic activities were measured in vulnerable and stable plaques collected from human atherosclerotic carotid arteries. Ex vivo affinity of P947 for metalloproteinases in vulnerable lesions was subsequently determined. Interaction between P947 and major cell types present in atherosclerotic plaques was also investigated in different cell lines: PMA-1-differentiated THP-1 (macrophage), Ox-LDL-treated THP-1 (foam cell), Jurkat cell line (lymphocyte), and human umbilical vein endothelial cell (HUVEC, endothelial cell). Molecular targeting of P947 was confirmed by fluorimetry, ICP-MS, and in vitro MRI approaches. Potential application of P947 for detecting atherosclerotic plaques by in vivo MRI was tested in a rabbit model of atherosclerosis. In vitro, P947 displayed affinities for purified MMPs, ACE, ECE-1, NEP, APA, and APN in the micromolar range. Interestingly, MMPs, ACE, and APN exhibited higher activities in vulnerable plaques from human atherosclerotic carotid samples, as compared to stable plaques. ECE-1, NEP, and APA had either no activity or the same low activity in both vulnerable and stable plaques. P947 showed micromolar affinities for MMPs, ACE, and APN secreted by plaque samples. Moreover, P947 bound to THP-1 macrophages and THP-1 foam cells in a concentration-dependent manner and with a higher intensity than the control contrast agents DOTA-Gd or P1135 (DOTA-Gd coupled to a scrambled peptide). In THP-1 macrophages, P947 inhibited largely (70%) and almost completely (95%) MMP and APN activities, respectively, which strongly suggested an MMP- and APN-dependent binding of P947 to these cells. This enzyme-specific binding was confirmed with in vitro MRI. Indeed, the T1 value of THP-1 cells decreased from 2.094 s (macrophages w/o P947) to 2.004 s (macrophages with 1 mM of P947). In addition, the Gd content measured by ICP-MS was 11.01 ± 1.05 fg Gd/macrophage when cells were incubated in the presence of P947 and only 5.18 ± 0.43 fg Gd/macrophage with the control product P1135. The difference of Gd concentration between both contrast agents corresponded to a specific accumulation of 5.83 fg Gd/cell, which may be detected by MRI. MR imaging in the atherosclerosi...

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Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation

Cited by 5 publications

References 47 publications

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis

Molecular and Cellular Targets of the MRI Contrast Agent P947 for Atherosclerosis Imaging

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