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 of binding to ALK2. It would also be useful to know how ALK2 inhibitors that bind TGFBR1 as well, do so structurally, so we can select away from any chemical motifs that are contributing to ALK5 binding.

Thus, I purified ALK5 and FKBP12 and set up 25 96-well crystal plates with ALK5 bound to FKBP12 (to stabilise it) with 5 different compounds and 5 different coarse screens, as detailed in my Zenodo upload here.

Two weeks later I still don’t have any hits in any of those plates.

Additionally, all the ACVR1 + compounds crystals that I thought were ok were really not ok when I sent them to the synchrotron. They were tricky to mount, as they fell into tiny pieces as soon as I touched them with the loop to try and mount them, and those that I did manage to mount diffracted poorly. They are not single crystals, but multiple crystals all squished and growing together in a clump. So, I need to purify more ACVR1 as well as the ALK5/FKBP12 complex and set them up with some more compounds, of which we have no lack.

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