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 …

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 …

Optimising an assay to detect alkaline phosphatase activity in C2C12 cells

I don’t just want to check whether the compounds I’m testing kill DIPG cells, I want to check that they’re doing this by altering BMP signalling too. However, I can’t do this with the DIPG cells, because if a compound does work, and kill them, there’ll be no cell left to actually measure the amount Read More …

Determining the EC50 of Tracer-6908 with ACVR1/ALK2-NanoLuc

In nanoBRET assay, a fluorescently labelled tracer binds to the ATP-binding pocket of ACVR1/ALK2. This binding brings the tracer into the proximity (<10nm) of Nano-Luciferase which is fused to the C-terminus of ACVR1/ALK2. Bioluminescence resonance energy transfer (BRET) occurs where the energy released by Nano-Luciferase is transferred to the fluorophore via intermolecular forces. Nano-Luciferase and Read More …

Purification and co-crystallisation of TGFBR1 (ALK5) and FKBP12 with five ATP-competitive ALK2 inhibitors – so much disappointment

As you are probably aware by now, if you’ve read a few of my blogs, we are testing ATP competitive inhibitors to inhibit ALK2 but not any of the other 6 ALK family members. Thus, most of our activity and cellular assays test ALK2 as well as ALK5 activity in order to check the specificity Read More …

Attending the first ever CRUK London Brain Tumour Conference

Hello there! This week I won’t be filling you in on my latest experiment, because instead I spent three days at the first ever Cancer Research UK Brain Tumour Conference held in the Royal Society of Medicine in London! (Also the biggest conference I’ve been to so far). The conference gathered over 30 great speakers Read More …

Determining the suitability of C2C12 and HEK293 in dual luciferase assay (DLA) for ACVR1 (ALK2) and TGFBR1 (ALK5)

Aside from having high potency towards ACVR1/ALK2, inhibitor compounds ideally should be highly selective and not target other members of the Transforming Growth Factor beta (TGFb) superfamily. TGFBR1/ALK5 is selected for off-target screening because of its potential role in cardiac functions. Firefly luciferase is transcribed downstream of ACVR1/ALK2 in BRE reporter plasmid and downstream of TBFBR1/ALK5 in CAGA reporter plasmid. Renilla luciferase is constitutively transcribed Read More …