Merck

A Generic Platform for Cellular Screening Against Ubiquitin Ligases.

Scientific reports (2016-01-09)
Timurs Maculins, Nikki Carter, Thierry Dorval, Kevin Hudson, J Willem M Nissink, Ronald T Hay, Husam Alwan
ABSTRACT

Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - low glucose, With 1000 mg/L glucose, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
RPMI-1640 Medium, Modified, with sodium bicarbonate, without L-glutamine and phenol red, liquid, sterile-filtered, suitable for cell culture