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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

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Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins.

Silvia Scarabelli^1,2, Kui Thong Tan^1,2, Rudolf Griss^1,2

¹École Polytechnique Fédérale de Lausanne , Institute of Chemical Sciences and Engineering, Lausanne CH-1015, Switzerland.

|August 3, 2017

Summary

This summary is machine-generated.

We developed a novel fluorescent biosensor method to measure drug uptake in living cells. This approach ranks drug candidates by assessing their binding to cellular targets, aiding drug discovery.

Keywords:

FRET-based sensor protein HCAII cellular absorption cellular clearance intracellular concentration p53-HDM2 interaction peptide therapeutics

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Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

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Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

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Published on: March 16, 2017

Area of Science:

Biochemistry
Cell Biology
Pharmacology

Background:

Accurate assessment of cellular drug uptake is crucial for drug discovery and development.
Existing methods for measuring intracellular drug concentrations can be complex and indirect.

Purpose of the Study:

To introduce a novel fluorescent biosensor approach for evaluating drug and drug candidate uptake into living cells.
To enable quantitative ranking of cellular uptake and intracellular concentrations of various compounds.

Main Methods:

Developing fluorescent biosensors by converting protein targets into measurable reporters.
Utilizing live-cell measurements of compound binding to cognate biosensors.
Comparing live-cell binding data with in vitro measurements to determine cellular uptake.

Main Results:

Demonstrated successful evaluation of cytosolic uptake for two distinct classes of inhibitors.
Showcased two sensor designs: a SNAP-conjugated inhibitor sensor for human carbonic anhydrase II inhibitors and a label-free sensor for p53-HDM2 protein-protein interaction inhibitors.

Conclusions:

The developed fluorescent biosensor strategy provides a robust method for assessing drug cellular uptake.
This approach facilitates the ranking of drug candidates based on their intracellular concentrations and target engagement.