A Universal Assay for Aminopeptidase Activity and Its Application for Dipeptidyl Peptidase-4 Drug Discovery

Bazhin, Arkadiy A.; Chambon, Marc; Vesin, J.-M.; Bortoli, Julien; Collins, James W.; Turcatti, Gerardo; Chou, Chieh Jason; Goun, Elena A.

doi:10.1021/acs.analchem.8b04672

Cited by 5 publications

(5 citation statements)

References 40 publications

(56 reference statements)

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“…In this part, d -cysteine is “caged” with DPP-4 specific recognition sequence of amino acids (Gly-Pro), resulting in a tripeptide Gly-Pro- d -Cys (GPc). Proteolytic activity of DPP-4 on this peptide enables the release of d -Cys and subsequent in situ formation of d -luciferin with HCBT . DPPIV is a transmembrane glycoprotein with proteolytic activity that is capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position. − The DPPIV expression level variation was associated with various pathological processes, especially in diabetes and malignant tumors, implying that DPPIV was a potential biomarker for the early disease diagnosis and treatment .…”

Section: Resultsmentioning

confidence: 99%

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

et al. 2021

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The abnormally expressed peptidases in human tissues are associated with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiology elucidation and early clinical diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, respectively, by simply adding l-leucine and Gly-Pro dipeptides at the N-terminus of d-cysteine, which were specifically controlled by peptidase cleavage and released free d-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, respectively. A good linearity between BL and peptidase was obtained in the concentration range of 2.5–40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0–40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, respectively. Additionally, approximately 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, especially in primary tumors.

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Section: Resultsmentioning

confidence: 99%

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

et al. 2021

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“…The d -Cys moiety can be readily conjugated to existing Cys residues or introduced at the C-terminus or the N-terminus of the peptide chain, providing great flexibility and applicability of our method to a wide range of therapeutic peptides or proteins. To increase assay sensitivity, our efforts focused on lowering the high background noise resulting from endogenous l -Cys. , Despite the fact that a considerable number of studies have utilized the split luciferin approach for in vitro and in vivo applications, ,,, this problem had not yet been addressed . We found that quenching the background signal with excess CBT prior to treatment with SLP yields remarkable signal-to-noise ratios, with signals up to 60-fold greater than background levels even at low nanomolar concentrations of the peptide.…”

Section: Discussionmentioning

confidence: 98%

“…To increase assay sensitivity, our efforts focused on lowering the high background noise resulting from endogenous L-Cys. 28,48 Despite the fact that a considerable number of studies have utilized the split luciferin approach for in vitro and in vivo applications, 17,32,49,50 this problem had not yet been addressed. 50 We found that quenching the background signal with excess CBT prior to treatment with SLP yields remarkable signal-to-noise ratios, with signals up to 60-fold greater than background levels even at low nanomolar concentrations of the peptide.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…Thus far, this approach has been successfully used for imaging and quantification of the activity of various proteases, such as caspase 3/7, caspase 8, and dipeptidyl peptidase 4 (DPP-4), both in vitro and in vivo. 25,30,32,33 However, this novel tool has never been used to assess the cellular uptake of biomolecules.…”

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confidence: 99%

“…This reaction occurs efficiently in various physiological solutions as well as in living cells and animals. ,,, Similar to caging strategies that have been previously reported for the full d -luciferin scaffold, the split luciferin reaction provides the opportunity to cage one or both of the reaction components (i.e., d -Cys and/or CBT). Thus far, this approach has been successfully used for imaging and quantification of the activity of various proteases, such as caspase 3/7, caspase 8, and dipeptidyl peptidase 4 (DPP-4), both in vitro and in vivo . ,,, However, this novel tool has never been used to assess the cellular uptake of biomolecules.…”

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confidence: 99%

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Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

et al. 2019

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Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel “Split Luciferin Peptide” (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

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Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals

et al. 2021

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Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a “portable bioluminescent” (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.

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A Universal Assay for Aminopeptidase Activity and Its Application for Dipeptidyl Peptidase-4 Drug Discovery

Cited by 5 publications

References 40 publications

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals

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