Analysis of fragments that bind to HAO1

In my previous post, I introduced our fragment screening results on HAO1. Specifically, I told you that we identified five promising fragment hits, from a little over 400 collected structures, clustering to three biologically relevant sites: the active site (fragments 1 and 2), the gating loop (fragment 5) and the subunit interface (fragments 6 and Read More …

Complex formation of FKBP12/BMPR1A and FKBP12/BMPR1B and subsequent crystallisation.

Having previously purified BMPR1A (Alk3) and BMPR1B (Alk6), I spent some time purifying FKBP12 to form complexes with each of the receptors. FKBP12 binds to BMPR1A and BMPR1B via a similar GS domain to that seen in ACVR1, on the N-terminal side of the kinase domain and may help stabilize the structures and thus help 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 …

Generating HTT Knockdown Models

The experimental set-up and results covered in this post can be found here: doi.org/10.5281/zenodo.1420537 I was previously using an HTT overexpression model to look at any changes in proteins related to HTT or its function. Unfortunately, none of the related proteins I looked at showed any significant change in protein levels with HTT overexpression. There Read More …

Screening Compounds against USP5 Zf-UBD with a 19F NMR assay #2

I used a medium-throughput 19F NMR assay to screen my second batch of compounds against USP5 zinc finger ubiquitin binding domain (Zf-UBD). To recap, the 19F NMR assay detects ligand binding by measuring perturbations in the resonance of a fluorinated tryptophan at the binding pocket. I selected the compounds for my second screen from SGC’s in Read More …

Determining the dual luciferase ALK5 IC50 values of 30 legacy ACVR1/ALK2 inhibitors

A large number of ACVR1/ALK2 inhibitors were previously synthesised by Paul Brenner’s team (Target Discovery Center, University of Oxford) for the purpose of treating Fibrodysplasia Ossificans Progressiva (FOP). Although these compounds were not designed with blood-brain-barrier permeability in mind, they can serve as good bench-marks for my cellular assays. Therefore, 30 of these legacy compounds Read More …

Speaking at the annual conference of the Society for the Study of Inborn Errors of Metabolism (SSIEM)

Hello again! This week I am in sunny, sunny Athens attending the SSIEM annual conference that brings together renowned experts in inborn errors of metabolism from around the world to present their latest pre-clinical and clinical research. I was fortunate enough to receive a generous travel grant from the SSIEM to give a talk this Read More …

Identifying fragments that bind to HAO1 by x-ray crystallography

In this post, I would like to share my recent work on HAO1 fragment screening by crystallography, as a means to identify inhibitor starting points. So why fragment screening by x-ray crystallography? Well there are two components to my method of choice: Fragments instead of complex compounds provide good starting chemistry Fragments are less than Read More …