Label Free ubiquitin assay system

Here is a new report about ubiquitin pathway system.
https://doi.org/10.1016/j.chembiol.2018.06.004
Recently ubiquitin system is becoming attractive drug targets. Known assay systems such as ELISA and SDSPAGE has limitation for through put and FRET is depended on the fluorescent.
The author developed and reported new HTS system to overcome the issue.

They use MALDI-TOF (rapifleX) with N15-labeled ubiquitin for the assay. N15-labeled ubiquitin is used for internal standard.
rapifileX
https://www.bruker.com/products/mass-spectrometry-and-separations/maldi-toftof/rapiflex-maldi-tissuetyper/overview.html
This assay system detects consumption of mono-ubuquitin directly.

To detect the covalent binder that binds to cys in ubiquitins, they recommends to use (tris(2-carbox- yethyl)phosphine instead of DTT or beta-mercaptoethanole( BME) for conducting the enzymatic reaction. And they conduct the assay with high ATP concentration to reduce the likelihood of identifying ATP analogs as inhibitors.
Well-designed system.

They performed HTS assay with 1430 FDA approved compounds as library and MDM2, ITCH and HOIP as E3 ligase.

Finally they got some hit compounds.
resveratrol showed week UBE1/UBE2L3/HOIP pathway inhibition activity. Hmm…..
bendamustine shows high potency.
https://en.wikipedia.org/wiki/Bendamustine
It seems irreversible binder I think.

Label free high though put assay is useful to reduce the risk of false positive and analyze ligand-protein interaction directly.
Technology and science is moving so fast….

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Published by iwatobipen

I'm medicinal chemist in mid size of pharmaceutical company. I love chemoinfo, cording, organic synthesis, my family.

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