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In a previous post, I described a ubiquitin-rhodamine110 (UbRho110) assay, and showed that a ZnF-UBD ligand antagonizes USP5 catalytic activity. UbRho110 is not a native substrate, and validation with native polyubiquitin species in an orthogonal assay is required. To address this, I developed and optimized an internally quenched fluorophore pair (IQF) assay. You can see Read More …
Structure Image Smiles Mol. Weight Cc1cc(C(=O)NCC(=O)O)ccc1S(=O)(=O)N1CCC(N2CCCC2)CC1 409.5 In a previous post, I showed the expansion of UBTR012574 chemical series from commercial sources- Enamine’s REAL database and Scifinder to shed some insight into the structural activity relationship (SAR) (Figure 1). Figure 1. UBTR012574 chemical series UBTR020988a which has an amide resulted in weaker potency for USP5 Read More …
Hi Here are the details of the assay I developed to measure interaction between USP5 and ubiquitin in cells. Ubiquitin specific peptidase 5 (USP5) belongs to the largest ubiquitin-specific protease (USP) subfamily and has a zinc finger at its N-terminal portion and two ubiquitin-associated domains in the large catalytic region that mediate polyubiquitin binding (PMID:28923280, Read More …
Hi all! Hope everyone is staying safe and practising social distancing during these difficult times. Working from home, I’ve decided to catch up on my open notebook. In a previous post, I purified various full length USP5 constructs and determined the binding affinity of ubiquitin to these constructs. I wanted to determine the stoichiometry of Read More …
In a previous post, we solved the co-crystal structure of the USP5 zinc finger ubiquitin-binding domain (ZnF-UBD) with a promising compound, UBTR012574, which had a potency of approximately 10 µM (Figure 1). W expanded the chemical series of UBTR012574 by docking analogue compounds from the Enamine REAL database and Scifinder. We decided to order 17 Read More …
A Happy New Year to all! I am back from vacation and ready to jump back into my work with USP5. Great news- we solved the co-crystal structure of USP5 with a promising compound, UBTR012574a (Figure 1) (discussed in my last post), just before the Christmas holidays. You can check out details about co-crystal growth, Read More …
If you’ve been following along my open notebook journey over the last two years, you’ll be excited to hear I’ve published my work on the first antagonists of USP5 ZnF-UBD in a peer-reviewed journal. Check it out here! In a previous post, I outlined my selection of the next set of analogues in my chemical Read More …
Today’s post describes testing if ZnF-UBD ligands are able to antagonize USP5 catalytic activity using a ubiquitin-rhodamine110 (UbRho110) assay, which was developed and optimized by Leanna Smith of the Biophysics group at SGC Toronto. You can see details on Zenodo. The UbRho110 assay is a fluorescence intensity assay that monitors the enzymatic activity of deubiquitinating Read More …
In a previous post, I described the expression and purification of full length (FL) USP5 constructs and also determined that zinc finger ubiquitin-binding (ZnF-UBD) ligands are able to displace ubiquitin in both the ZnF-UBD and the FL protein in a fluorescence displacement assay. In today’s post, I describe using a surface plasmon resonance (SPR) assay Read More …
Hi all, today’s post covers some concepts that I have previously described: expression and purification of protein constructs, and testing ligands in a displacement assay. If you’ll recall, I tried to develop and optimize a fluorescence polarization (FP) displacement assay for the ZnF-UBD of USP5 for some time but found that FP was a no Read More …
