Journal: British Journal of Cancer

Article Title: UCHL1 as a novel target in breast cancer: emerging insights from cell and chemical biology

doi: 10.1038/s41416-021-01516-5

Figure Lengend Snippet: a Potent UCHL1 inhibitors, ABPs and less specific compounds with potential off-targets; IC 50 were determined by monitoring cleavage of rhodamine110 from a ubiquitin substrate (Ub-Rho) for compound 1 and 2 , and by cleavage of Rho-morpholine from Ub-Rho-morpholine substrate for 6RK71 and 8RK59 using fluorescence intensity assay; . b Structures of highly selective and potent UCHL1 inhibitor 3 (( S )-enantiomer) and ABP IMP-1710 (( S )-enantiomer) and structurally similar inactive inhibitor IMP-1711 (( R )-enantiomer, negative control); c Promiscuous and moderately potent UCHL1 inhibitor LDN-57444 . IC 50 of 3 , IMP-1710 , IMP-1711 and LDN-57444 were determined by a fluorescence polarisation assay using a ubiquitin substrate Ub-Lys-TAMRA .

Article Snippet: In parallel, Panyain et al. in collaboration with Mission Therapeutics reported high quality, low nanomolar activity UCHL1 small-molecule probes and inhibitors, which target UCHL1 with minimal off-target activity, providing the most potent and selective tools currently available to probe UCHL1 in cancer [ ].

Techniques: Ubiquitin Proteomics, Fluorescence, Negative Control

Journal: British Journal of Cancer

Article Title: UCHL1 as a novel target in breast cancer: emerging insights from cell and chemical biology

doi: 10.1038/s41416-021-01516-5

Figure Lengend Snippet: Schematic representation of a measurement of target (UCHL1) engagement by gel fluorescence and western blotting using ABPs. Generally, ABP labelling is directed towards a particular target protein or protein class. ABPs may contain latent handles such as an alkyne, which may be bioorthogonally ligated with a reporter such as a fluorophore or a biotin affinity tag. If a fluorophore is used, labelled proteins may be separated by SDS-PAGE and directly visualised using fluorescence scanning, while biotinylated target proteins are generally enriched on a neutravidin-immobilised resin and visualised on western blot using target-specific antibodies; b quantification of on- and off-targets using ABPs and competitive mass spectrometry (MS)-based proteomics. Cells are treated with selective inhibitors/drugs, followed by intracellular labelling with ABPs, lysis, bioorthogonal ligation to biotin and enrichment of biotinylated proteins on Neutravidin-agarose resin. On-resin digestion generates peptides from probe-labelled proteins, which are subsequently analysed using liquid chromatography-mass spectrometry. Differential labelling in inhibitor-treated and untreated samples are quantified, enabling the identification of novel drug targets as well as sites of protein modification for further drug development; c Imaging target protein activity in live cell or in in vivo (e.g. zebrafish embryo) cancer models using fluorescently labelled ABPs and fluorescence microscopy.

Article Snippet: In parallel, Panyain et al. in collaboration with Mission Therapeutics reported high quality, low nanomolar activity UCHL1 small-molecule probes and inhibitors, which target UCHL1 with minimal off-target activity, providing the most potent and selective tools currently available to probe UCHL1 in cancer [ ].

Techniques: Fluorescence, Western Blot, SDS Page, Mass Spectrometry, Lysis, Ligation, Liquid Chromatography, Modification, Imaging, Activity Assay, In Vivo, Microscopy