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  • Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery.

Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery.

Scientific reports (2015-03-13)
Hugo M Botelho, Inna Uliyakina, Nikhil T Awatade, Maria C Proença, Christian Tischer, Lalida Sirianant, Karl Kunzelmann, Rainer Pepperkok, Margarida D Amaral
ABSTRACT

Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.

MATERIALS
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Product Description

Supelco
Discovery® C8 Supelguard Guard Cartridge, 5 μm particle size, L × I.D. 2 cm × 4 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 3 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 5 cm × 4.6 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 5 cm × 2.1 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 2.1 mm
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Discovery® C8 Supelguard Guard Cartridge, 5 μm particle size, L × I.D. 2 cm × 4 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 12.5 cm × 4 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 12.5 cm × 4.6 mm
Supelco
Discovery® C8 Supelguard Guard Cartridge, 5 μm particle size, L × I.D. 2 cm × 2.1 mm
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
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Discovery® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm