Purification of GS loop mutants of ALK2

…and analysing their activity! Following on from the work I talked about here I’ve been able to start looking at the influence the different phosphorylation sites have over the ability of ALK2 to activate SMAD1. Just coming through the SGC expression pipeline are several constructs containing mutations in various combinations of the sites known to Read More …

Different methods to evaluate the effects of M4K compounds on DIPG patient-derived cell lines

Background: Aside from determining the optimal numbers of DIPG cells to be seeded for viability assay, it is also important to validate that measurements using CellTiter Glo are in agreement with other methods of determining cell viability. Experimental design: I chose to compare the EC50 values of M4K2009 on HSJD-DIPG-007 when measured using different methods. Read More …

Shockingly effective – electroporation of ribonucleoprotein complexes into DIPG cells

As soon as possible I’m hoping to use CRISPR/Cas9 genetic editing to make sets of DIPG patient cells that differ only in one gene (either with/without the whole gene or with/without mutations of interest). This will allow me to make far more accurate comparisons of behaviour between the cells in relation to specific genes or Read More …

Selectivity against, and action of, TNIK kinase in relation to ALK2.

Sorry I’ve not written for a while. I had some annual leave to use up and then I’ve been helping out with a residential EMBO course that was being co-organised by one of the group leaders in my department. The course was an introduction to a wide variety of biochemical and biophysical techniques. I was Read More …

Optimisation of viability assay for DIPG patient-derived cell lines

Background: Evaluation of the efficacy of M4K compounds in DIPG patient-derived cell lines is essential before any promising compounds can be further tested in mouse xenograft models. This approach can aid in narrowing down clinical compound candidates and reduce the time, resources and animal sacrifice needed downstream. A robust and efficient readout for the changes Read More …

Synthesis of promiscuous ALK2 inhibitors-continuation-2

Continuing with our goal to synthesize three compounds as ALK2 inhibitors, let’s refer back to Scheme 2 in the previous post on this topic, compounds 6, 8 and 9 were synthesized in good yields (65-90%), however compound 10 could not be obtained.   Different conditions were tested to induce the cyclization of the bromo-pyrazine-2-carboxamide 9 Read More …

ACVR1 Crystals taken to the Diamond Light Source (Beamline I04.) – no data sets collected.

Following up on the crystals from my last post I sent them along on the last Diamond trip we had to beamline I04 but regrettably didn’t get any usable datasets. Perhaps fortunately there is a possible explanation for this that isn’t just ‘the crystals were a bit too rubbish’. We send our crystals to the Diamond Read More …

How many cells in DIPG behave like stem cells?

Stem cells are a set of cells that, unlike most cells in the body, can divide as many times as they want, and can turn into any cell in the body. The mutations in cancer often make the cells behave more like stem cells, because in order to grow into a large tumour they may Read More …

Re-Purification of ALK2 for crystallisation with different compounds from M4K.

I’ve been lucky enough to have a student (Mira) in the lab working with me for the past few weeks and so I’d like to write about the work that we’ve been doing together. We spent some time cleaning up an old sample of ALK2 that Ros had left over in the freezer to set Read More …

The Benefits of Conference attendance: BMP Signalling in Cancer II.

I’ve got a few things to catch up with on the blog – one of them is to talk about a conference I attended in Oxford recently. I attended the Biochemical Society BMP Signalling in Cancer II meeting which was held in St. Annes College, Oxford. At this meeting I presented a poster on the Read More …