Short form TGFBR1 (ALK5) purification and co-crystallisation with M4K2006 and M4K2089

In the absence of our “workhorse” protein, ACVR1 residues 208-499 (constitutively active Q207D mutant) (as we are still trying to work out why it won’t express at the moment), and the complete lack of crystals/diffracting crystals for the TGFBR1/FKBP12 complex, I have shifted to some other constructs we have to keep things moving. I had Read More …

Purification and crystallisation of ALK2 (R206H mutation) with M4K Pharma compounds.

As part of an ongoing collaboration with Meds4Kids Pharma (M4KPharma), we are testing a range of compounds identified and generated by them as being of interest in developing a molecule specific against ALK2 that has the right properties help treat some cases of DIPG. This also has significant implications for development of a drug that Read More …

Still no protein, and no diffraction from TGFBR1/FKBP12 crystals, but outreach is fun!

I’m sad to report that a couple of test expressions later, we’re still no wiser as to why ACVR1 isn’t expressing. It’s worked on a couple of small scale expression test plates, but just doesn’t want to scale up anymore. We may be looking at having to remake the virus. In the meantime, I had Read More …

Things still not going according to plan …

I ended last time saying Shubhash had saved the day by finding a better virus stock and growing up some more cells for me. Sadly, this did not work – still no expression. So we tried halving the amount of virus used, as using too much can infect too many cells leaving not enough making Read More …

Determining the nanoBRET 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 …

Determining the IC50 of M4K1062 with ACVR1/ALK2-NanoLuc and different concentrations of Tracer-6908

The EC50 of Tracer-6908 with ACVR1-c-nanoLuc and ideal conditions for the target engagement assay have been determined in a previous experiment. However, it is still necessary to verify that the IC50 values determined in the assay are closed enough approximation to the actual IC50 values of the compounds. If the IC50 values are strongly influenced Read More …

Pull down of XIAP with type I & II receptors. Tm shifts of M4K Pharma compounds with Alk2, Alk5 and RIPK2.

XIAP and its interaction with type I and type II receptors. Having spent time over the past few weeks purifying all the components needed for a pull down with XIAP, I did the experiment that looked for interactions between XIAP and various type I and type II receptors in both the un-phosphorylated and phosphorylated form. Read More …

A bad day in the lab – science never goes as expected!

I promised to post again today with an update on in-house screening of my hit for fragment screening, but sadly, there’s nothing to update, as yesterday turned into a litany of disappointment (not to be too much of a drama queen!). First, I started a fresh purification of ACVR1 to prepare for setting up new Read More …

Various purifications, and a few crystal hits

Gosh, it’s been a while since I posted – not because I have nothing to report, but because I’ve been super busy in the lab trying to get things done, as well as having loads of meetings and a symposium. So, in the meantime, I’ve done two purifications of ACVR1 and set up crystal plates, Read More …