Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

Zhang, Ming; Chakraborty, Subhasish; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

doi:10.1172/jci81086

Cited by 27 publications

(33 citation statements)

References 53 publications

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“…4C and 4D) and NT-mediated β-arrestin2-GFP recruitment using a translocation assay (fig. 4E) 43-45 . In contrast to the binding results, where ML314 increases receptor Bmax, the maximal absolute amount of endocytosed receptor is unchanged from that observed with NT alone (fig.4C).…”

Section: Resultsmentioning

confidence: 99%

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse

et al. 2016

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Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, β-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J wild type mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.

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Section: Resultsmentioning

confidence: 99%

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse

et al. 2016

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“…FAP-fluorogen pairs can be selected based on binding affinities and fluorescent spectra suited for specific applications. Fluorescence generated from dL5-MG can be detected optically in superficial tissues, but other FAP systems, such as far-red fluorogenic dyes currently being explored, may be used for internal imaging [62]. There are also promiscuous FAPs that can bind more than one fluorogen [63], with alternate excitation and emission wavelengths and varying affinity constants for fluorogen binding.…”

Section: Discussionmentioning

confidence: 99%

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

Liu

Saunders

Bagia

et al. 2016

Journal of Controlled Release

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Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a β-fibrillizing peptide (βFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a βFP that forms β-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose–response titration experiments showed that loading of IgG into the film was mediated by pAGMG bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAGMG after five days, comparedto close to 90% in films without pAGMG. Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAGMG. When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

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“…Cell‐excluded and permeable to the mammalian PM. MG2p and MG‐Ester have been used successfully with Mars1Cy and Mars1 FAPs…”

Section: Fluorogen Activating Protein Platformmentioning

confidence: 99%

“…The dL5** and AM2‐2 FAP tags noncovalently bind MG and TO1 dye derivatives respectively with high affinities (Figures and ). Later on other scFv tags were identified and characterized for binding and activation of dimethylindole red (DIR) and oxazole thiazole‐blue (OTB) fluorogenic dyes, and near‐infrared dyes, SKC1 and SKCi1 (Figure ) . FAP‐dye binding rigidizes the chemical structure, suppressing some bond‐rotations responsible for internal conversion and nonradiative relaxation in the electronic excited state, resulting in increased fluorescence emission.…”

Section: Fluorogen Activating Protein Platformmentioning

confidence: 99%

“…Later on other scFv tags were identified and characterized for binding and activation of dimethylindole red (DIR) and oxazole thiazole-blue (OTB) fluorogenic dyes, and near-infrared dyes, SKC1 and SKCi1 ( Figure 5). 23,[28][29][30] FAP-dye binding rigidizes the chemical structure, suppressing some bond-rotations responsible for internal conversion and nonradiative relaxation in the electronic excited state, resulting in increased fluorescence emission. The first synthesized dyes, MG-2p, MG-11p and TO1-2p, possess an hydrophilic diethylene glycol diamine, carrying a positive charge and significant hydration that prevents these dyes from passively crossing the PM and entering the cell.…”

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confidence: 99%

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Fluorogen activating protein toolset for protein trafficking measurements

Perkins

Bruchez

2020

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Assessing protein trafficking in live cells is a core strength of fluorogen activating protein technology. The rapid and specific labeling of a tagged protein of interest, intracellular or surface, with a variety of fluorogenic dye derivatives creates a toolset suitable for scalable, dynamic multi‐color fluorescence experiments and direct quantitative trafficking‐related measurements in single cells and cell populations, using fluorometry, flow cytometry and microscopy.

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Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

Cited by 27 publications

References 53 publications

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

Fluorogen activating protein toolset for protein trafficking measurements

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