A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription, Translation, or Proteasomal Degradation

Abstract

Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability, and interfere with the processes of transcription and translation, or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multi-step processing, and are not easily amenable to high-throughput screening (HTS). The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high intensity light emitting diode (LED) array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.

Photochemical and Photobiological Activity of Ru(II) Homoleptic and Heteroleptic Complexes Containing Methylated Bipyridyl-type Ligands

“…55 Dendra2 is a fluorescent protein that is expressed with a chromophore that can be excited at 485 nm to induce emission in the green region of the spectrum at 530 nm, similar to GFP. However, the chromophore can be subjected to a photochemical reaction by exposure to 405 nm light, resulting in emission at 595 nm.…”

“…The analysis of the ratio of the two emission features allows for correction for cell number and cell health over the time course of the experiment. 55 …”

“…An in-cell assay we recently developed to monitor effects on protein production 55 gave intriguing results for the different compounds as a function of compound structure. The bis-heteroleptic compound 1 , containing the 4,4′, 6,6′-tetramethyl-2,2′-bipyridine ligand was able to completely abrogate protein production at high concentrations when irradiated, but its tris-homoleptic analogue 4 was even more potent, with effects observed at the 30 μM concentration point (Figure 2C and D).…”

Photochemical and Photobiological Properties of Pyridyl-pyrazol(in)e-Based Ruthenium(II) Complexes with Sub-micromolar Cytotoxicity for Phototherapy

“…To investigate the impact of the Ru(II) complexes on the essential biological processes, a cell-based transcription and translation assay was performed using the photoconvertible protein, Dendra2, as a reporter for protein synthesis. 75 This allowed for a real-time report in live cells of any damage to the DNA, RNA, or the ribosome or inhibition of any essential components of the cellular machinery responsible for the processes of transcription and translation.…”

Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes

“…To discriminate between inhibition of protein production and induction of cell death, we used a photoconvertible protein, Dendra2, as a reporter, since it is able to provide information on both aspects of cell viability and new protein production simultaneously. 49 Dendra2 undergoes a photochemical reaction, transforming from a green fluorescent protein to a red fluorescent protein when exposed to 405 nm light. The photoconverted “Red” Dendra2 emission provided a stable internal reference for cell health and cell number for all samples, while new protein production (after light exposure) is reflected in the “Green” Dendra2 emission.…”