Intramolecular and Intermolecular FRET Sensors for GPCRs – Monitoring Conformational Changes and Beyond

Kauk, Michael; Hoffmann, Carsten

doi:10.1016/j.tips.2017.10.011

Cited by 53 publications

(43 citation statements)

References 71 publications

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“…Finally, the last few years have been characterized by the application of a variety of fluorescence-based methods for GPCR structure biology and drug discovery [17]. These techniques involve the introduction of a fluorescent tag on a GPCR or on a GPCR ligand, leading to fluorescent ligands, which are discussed here [9,[18][19][20]. The aim of this review is to give a panorama of the available chemical probes for the ARs to researchers working in this field or medicinal chemists working on ARs or other GPCR targets.…”

Section: Introductionmentioning

confidence: 99%

Chemical Probes for the Adenosine Receptors

2019

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Research on the adenosine receptors has been supported by the continuous discovery of new chemical probes characterized by more and more affinity and selectivity for the single adenosine receptor subtypes (A1, A2A, A2B and A3 adenosine receptors). Furthermore, the development of new techniques for the detection of G protein-coupled receptors (GPCR) requires new specific probes. In fact, if in the past radioligands were the most important GPCR probes for detection, compound screening and diagnostic purposes, nowadays, increasing importance is given to fluorescent and covalent ligands. In fact, advances in techniques such as fluorescence resonance energy transfer (FRET) and fluorescent polarization, as well as new applications in flow cytometry and different fluorescence-based microscopic techniques, are at the origin of the extensive research of new fluorescent ligands for these receptors. The resurgence of covalent ligands is due in part to a change in the common thinking in the medicinal chemistry community that a covalent drug is necessarily more toxic than a reversible one, and in part to the useful application of covalent ligands in GPCR structural biology. In this review, an updated collection of available chemical probes targeting adenosine receptors is reported.

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Section: Introductionmentioning

confidence: 99%

Chemical Probes for the Adenosine Receptors

2019

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“…Considering receptor promiscuity between the AVT and isotocin peptides and receptors, it would be useful to determine whether isotocin plays a role in behavioral transformation in wrasses, i.e. promotes expression of courtship behavior typical of TP males, and, if so, whether it acts in part via binding to AVT receptors; Fluorescence resonance energy transfer (Kauk and Hoffmann, 2018) could be used to identify interactions between isotocin and AVT receptors.…”

Section: Discussionmentioning

confidence: 99%

Latency for facultative expression of male-typical courtship behaviour by female bluehead wrasses depends on social rank: The ‘priming/gating’ hypothesis

Price

Luong

Bell

et al. 2018

Journal of Experimental Biology

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Although socially controlled sex transformation in fishes is well established, the underlying mechanisms are not well understood. Particularly enigmatic is behavioural transformation, in which fish can rapidly switch from exhibiting female to male-typical courtship behaviours following removal of 'supermales'. Bluehead wrasses are a model system for investigating environmental control of sex determination, particularly the social control of sex transformation. Here, we show that the onset of this behavioural transformation was delayed in females that occupied low-ranking positions in the female dominance hierarchy. We also establish that expression of maletypical courtship behaviours in competent initial-phase (IP) females is facultative and gated by the presence of terminal-phase (TP) males. Dominant females displayed reliable TP male-typical courtship behaviours within approximately 2 days of the removal of a TP male; immediately following reintroduction of the TP male, however, females reverted back to female-typical behaviours. These results demonstrate a remarkable plasticity of sexual behaviour and support a 'priming/gating' hypothesis for the control of behavioural transformation in bluehead wrasses.

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“…The second known in vivo model visualized intramolecular changes after agonist and antagonist binding by introducing fluorophores into the third extracellular loop and the C terminus of different GPCRs. Upon activation, the distance between both fluorophores changes and an alteration in the energy transfer can be measured [105,106]. Initial experiments to transfer these energy transfer-based sensors were recently performed in CF systems [107].…”

Section: G-protein Coupled Receptors (Gpcrs) and Drug Discovery In Cementioning

confidence: 99%

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

et al. 2020

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Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. Key PointsCell-free protein synthesis has the potential to become an alternative method for the rapid and highly parallel expression of a diverse range of proteins.Cell-free systems utilize the translation machinery of the cells, bypassing the constraints of the cell membrane and thus offer openness and configurational flexibility even for the synthesis of difficult-to-express proteins.In comparison to traditional approaches, cell-free systems can be time-saving, from initial synthesis to downstream applications.

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Intramolecular and Intermolecular FRET Sensors for GPCRs – Monitoring Conformational Changes and Beyond

Cited by 53 publications

References 71 publications

Chemical Probes for the Adenosine Receptors

Chemical Probes for the Adenosine Receptors

Latency for facultative expression of male-typical courtship behaviour by female bluehead wrasses depends on social rank: The ‘priming/gating’ hypothesis

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

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