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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 …
Purification of proteins for a pull down experiment between XIAP and various type I and type II receptors. XIAP is known to bind to the kinase RIPK2. RIPK2 is one of the few kinase proteins that shows overlap of reactivity to some of the compounds that we’ve been looking at in the context of ALK2 Read More …
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 …
After the disappointment of looking through ~13000 crystal drop images over the past couple of weeks and finding pretty much nothing in most of them and nothing that actually diffracted nicely in the rest, it was incredibly heartwarming and hugely inspiring to attend the FOP family and friends conference in Manchester last weekend. FOP Friends is Read More …
Deposition: In the past two weeks my work has mostly been full of dealing with structures. The first thing I needed to do was deposit some structures of ALK2 co-crystallised with three drug molecules that I had solved for our collaborators Swen Hoelder and Liam Hudson at ICR, who had been developing compounds against ALK2. Read More …
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 …
One of the more unpredictable parts of crystallography is also one of the most fundamental – will the protein crystallise? Despite decades of work done on protein crystallisation, and numerous ‘tricks of the trade’ to maximise the chances, whether a protein will actually form a crystal is still relatively random. With ALK2 we’ve crystallised it Read More …
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 …
It’s been a little while since I posted. I’ve been really busy, but haven’t had that much to report. I’ve been doing some processing of the fragment screening data we have so far. We found one possible hit in the pocket we are looking at for allosteric ligands, which initially looked very exciting, but on Read More …
New compounds developed by Meds4Kids (M4K) and other collaborators against ALK2 would benefit from being crystallised so we can look at the structure of the complex and explain exactly how these compounds bind. This helps us work out how to improve them for both potency and selectivity and explain why some bind better than others. Read More …
