Purification of ACVR1 for co-crystallisation with new compounds and for fragment screening crystals

I was away on leave last week, but happily, before I left, I set up 25 crystal plates of ACVR1 with various compounds. The details of the protein purification, the compounds used and some of the crystals obtained are here, on Zenodo. So I was able to at least come back to something after my Read More …

Developing an assay of viability in DIPG cell lines

Over the past few weeks I’ve been perfecting a method for screening a small set of therapeutic compounds on DIPG cell lines. Before starting I made sure to discuss the techniques used in other groups to make sure that I design an assay that is comparable to theirs, although I may make my own changes Read More …

Update on fragment screening of ACVR1 co-crystallised with LDN-193189

I’ve put the Molprobity stats for the model I have refined to run my fragment screening datasets against in Zenodo. It looks pretty good, and I was happy when the parameters finally all went into the green. I have about 90 datasets with resolution lower than 2.6 Å, so hopefully I’ll get at least one Read More …

Transfectability of different patient-derived DIPG and Glioblastoma cell lines

Whether or not a cell line can be transfected efficiently is an important factor to consider when designing experiments. Often times scientists would like to deliver into the cells DNA or RNA with the instructions to produce specific proteins or to delete away specific proteins. Transfection via liposome (eg: Lipofectamine 2000) is a relatively simple Read More …

Determining whether anti-phospho-SMAD1/5 and anti-phospho-SMAD2 antibodies can recognize formaldehyde-fixed epitopes

Kinase activity of ALK2 and ALK5 in the cells can be quantified using specific antibodies that bind to target proteins once they are phosphorylated. However, conventional detection in Western blot requires large amount of samples and processing time (proteins are extracted from a large number of cells, linearized, separated in electric field and blotted onto a Read More …

Efficacy of inhibitor on wild-type ALK2 and R206H mutant in C2C12 cells (by DLA)

There are concerns that compounds that are effective in inhibiting ALK2 by occupying its ATP-binding pocket might have reduced efficacy against mutant ALK2. That will be undesirable since the compounds should also target the gain-of-function mutant ALK2 in DIPG cells. The following experiment takes advantage of the fact that Activin A activates ALK2-R206H mutant but Read More …

Recording the growth of a DIPG cell line

Repeatability is a cornerstone of science, but cells behaviour can change quite significantly if you aren’t careful when looking after them. To ensure that I have the right number of DIPG cells to complete an important experiment, and that they’re all growing healthily, I want to record how fast they grow, and at what density Read More …

Optimisation of ligand concentration for the activation of ALK2 and ALK5

ALK2 and ALK5 are Type I receptors on the cell surface that can be activated by specific ligands (illustrated in the diagram below). Once activated by ligand, ALK2 or ALK5 interact with Type II receptor to be phosphorylated (phospho-group chemically attached to the protein). Subsequently, ALK2 phorphorylates SMAD1/5/8 while ALK5 phorphorylates SMAD2. Phosphorylated SMADs then  Read More …

Optimising transfection of the C2C12 myoblast cell line

Within a month (hopefully) I’ll be convincing C2C12 myoblast cells to express all kinds of mutant ALK2 to test its activity and interactions with other proteins within the cell. I’ll be doing this by treating them so that they’ll take up pieces of circular DNA (plasmids) that express those mutant proteins (i.e. transfecting them). Seeing Read More …

Project overview: Establishing Cellular Assays to Screen for ALK2 Inhibitors

Diffuse Intrinsic Pontine Glioma (DIPG) is a type of brain tumour in the brainstem which is highly aggressive and occurs in children. Treatment options for DIPG are very limited because these tumours do not respond to the chemotherapy drugs currently available for adult gliomas. Whole genome and exome sequencing identified several frequently mutated genes in Read More …