Roncoroni C.,Top Transgenic Operative Products Srl |
Rizzi N.,Top Transgenic Operative Products Srl |
Brunialti E.,Top Transgenic Operative Products Srl |
Cali J.J.,Promega Corporation |
And 3 more authors.
Pharmacological Research | Year: 2012
Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs. © 2012 Elsevier Ltd. All rights reserved.
Cali J.J.,Promega Corporation |
Ma D.,Promega Corporation |
Wood M.G.,Promega Corporation |
Meisenheimer P.L.,Promega Biosciences |
Klaubert D.H.,Promega Biosciences
Expert Opinion on Drug Metabolism and Toxicology | Year: 2012
Introduction: The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase. Areas covered: This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production. Expert opinion: Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies. © 2012 Informa UK, Ltd.
Duellman S.J.,Promega Corporation |
Valley M.P.,Promega Corporation |
Kotraiah V.,Exonhit Inc. |
Vidugiriene J.,Promega Corporation |
And 6 more authors.
Analytical Biochemistry | Year: 2013
The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B = 64) and Z′ = 0.75. © 2012 Elsevier Inc. All rights reserved.
Robers M.B.,Promega Corporation |
Binkowski B.F.,Promega Corporation |
Cong M.,Promega Corporation |
Zimprich C.,Promega Corporation |
And 8 more authors.
Analytical Biochemistry | Year: 2015
Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. © 2015 Elsevier Inc.All rights reserved.
Takemoto K.,Hokkaido University |
Takemoto K.,Yokohama City University |
Matsuda T.,Hokkaido University |
McDougall M.,Promega BioSciences |
And 7 more authors.
ACS Chemical Biology | Year: 2011
Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (1O2) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of 1O2 than fluorescein and about 5-fold efficiency in CALI of â-galactosidase by using an eosin-labeled anti-â-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-ã and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells. © 2011 American Chemical Society.
PubMed | Promega Corporation and Promega Biosciences
Type: | Journal: Analytical biochemistry | Year: 2015
Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.