Testing Selectivity of USP5 Zf-UBD Analogues with SPR Assay

In a previous post, I tested hit analogues and found compound XSR00035795a (Figure 1) had increased potency and ligand efficiency; however, compound XSR00035795a was also found to bind to HDAC6 Zf-UBD, so it was not selective towards USP5. Figure 1. XSR00035795a We hypothesized that it was possible to extend the aliphatic group on the carboxylic Read More …

USP5 Zf-UBD Co-Crystal Structure with Compound XSR00035795a & Testing Selectivity

In a previous post, I tested hit analogues against USP5 Zf-UBD using a SPR assay. Compound AE-641/11456811, now aptly re-named XSR00035795a (an SGC global ID- since our database infrastructure is finally re-vamped), had a binding affinity of approximately 60 µM. The addition of a methyl group on the carboxylic chain of the compound increased potency Read More …

Testing Hit Analogues against USP5 Zf-UBD with SPR Assay #1

In a previous post, I described using hit expansion tools such as commercial substructure searches, docking, and free energy perturbation (FEP) simulations to explore the structure activity relationship (SAR) of the chemical series focused on the Zf-UBD of USP5. The first batch of commercially ordered compounds arrived just before the Christmas holidays. I decided to Read More …

Development of a Mass Spectrometry USP5 Catalytic Activity Assay

Now that I’ve got my biophysical screening assays developed (19F NMR, SPR), as well as my hit expansion campaign started for small molecule inhibitors against the USP5 Zf-UBD, I need to be able to test if these small molecule inhibitors will antagonize the catalytic activity of USP5. It has been speculated in literature that the Read More …

Hit Expansion using Substructure Search, Virtual Screening & Free Energy Perturbation

Molecular dynamic simulations are a powerful tool for modeling biomolecular systems such as ligand binding to proteins. In a previous post I was able to get co-crystal structures of ligands which bind to the USP5 zinc finger ubiquitin binding domain (Zf-UBD). In order to explore the structure activity relationship (SAR) of the chemical series, so Read More …

Screening Compounds of Interest against USP5 Zf-UBD by SPR #2

I used a surface plasmon resonance assay to determine the binding affinities of hit compounds that were identified in 19F NMR spectroscopy screens against the zinc finger ubiquitin binding domain of USP5. To see how these assays work, check out some of my previous posts! You can see details of the SPR experiment on Zenodo. Read More …

Developing a Tryptophan Fluorescence Assay for Screening Ligands against USP5 Zf-UBD

For those following the USP5 project on my open notebook, you will recognize that assay development has been quite a challenge for screening ligands against USP5 zinc finger ubiquitin binding domain (Zf-UBD). In the past, I was unsuccessful in developing a differential scanning fluorimetry, and fluorescence polarization screening assay. I then tried 19F NMR spectroscopy screening, Read More …