Bromodomains have nothing to do
with bromine. Rather, they are small (~110 amino acid) domains that recognize
acetylated lysine residues, a common modification on histones, and are thus key
epigenetic “readers”. Humans have more than 60 of them, and as you can imagine
selectivity is not assured. However, fragments have proven very useful in
targeting these proteins. Since the first mention of bromodomains on Practical Fragments back in 2011 the
number of posts has been growing rapidly, so for the first time ever we’ve
decided to devote an entire month to the topic.
In other words, July is
bromodomain month! We’ll start with two papers against the bromodomain BRD9,
part of the SWI/SNF chromatin remodeling complex that seems to be important for
acute myeloid leukemia.
The first paper, in J. Med. Chem. (and open access), is
published by Laetitia Martin and collaborators at Boehringer Ingelheim,
University of Oxford, and Cold Spring Harbor. The researchers used three orthogonal
biophysical screening methods: differential scanning fluorimetry (DSF), surface
plasmon resonance (SPR), and microscale thermophoresis (MST). A library of 1697
fragments was screened at 0.4 mM (DSF), 0.1 mM (SPR) or 0.5 mM (MST), and hits
were then validated using 15N HSQC NMR. The 77 hits that confirmed
were taken into crystallography, producing 55 structures.
Validation rates in the NMR
secondary screen were excellent for DSF (94%) and SPR (84%) but less so for MST
(31%). That said, of the 38 validated hits from MST, 29 were not found in
either of the other techniques, and 14 of these produced crystal structures.
This is a useful reminder that while screening cascades can whittle down many
hits, they do run the risk of throwing out the proverbial babies along with the
bathwater.
In parallel with the biophysical
screens, a virtual screen of ~73,500 fragments was conducted using Glide to
identify 208 fragments that were then tested using SPR and DSF. This led to 23
hits, 11 of which produced crystal structures.
Two of the more potent fragments
were the structurally related compound 3 (from the biophysical screen) and
compound 4 (from the virtual screen). Optimization started with compound 4 by
adding electron donating groups to the phenyl ring to try to improve a stacking
interaction observed in the crystal structure. This led to compound 10, and
building out the other ring to make it more similar to fragment 3 led to
BI-9564.
BI-9564 has low nanomolar
activity in both a biochemical assay as well as isothermal titration
calorimetry (ITC). It is also quite selective: among 48 other bromodomains, it
only hits the closely related BRD7 and CECR, and it is >10-fold more potent
on BRD9. None of a panel of 321 kinases were inhibited with IC50
< 5 µM, and only 2 of 55 GPCRs were inhibited. The compound is also cell
active, reasonably soluble, has good pharmacokinetics in mice, and orally
bioavailable. In short, BI-9564 is an excellent chemical probe – and is in fact
being offered as such.
While we’re on the subject of
BRD7 and BRD9, it’s worth noting another recent paper, this one in ChemBioChem from Ke Ruan and colleagues
at the University of Science and Technology of China. The researchers screened
their library of 890 fragments against BRD7 using three different
ligand-detected NMR techniques: STD, WaterLOGSY, and CPMG. Fragments were
screened in pools of 10 with each fragment present at 400 µM. This yielded just
10 hits, of which 5 confirmed when tested individually. Protein-observed NMR
was then performed on these, suggesting that they all bind in the acetyl-lysine
recognition sites; they have similar affinities for both BRD7 and BRD9, with
dissociation constants between 22 and 600 µM. Crystallography confirmed the
binding mode for one of the fragments bound to BRD9. Interestingly, this showed
quite a bit of plasticity in the protein compared to the un-liganded structure.
Indeed, the BI researchers suggest that different degrees of protein
flexibility between BRD7 and BRD9 could account for the selectivity differences
observed for BI-9564.
Stay tuned next week for more
fragment-screening against a different class of bromodomains!
2 comments:
I am really enjoying the recent burst of Bromodomain small molecule modulators popping up in the literature. It is chemical genetics in action! A recent paper in Nature Chemical Biology (PMID: 27376689) further highlights the power to selectively target these domains and sheds light into their cross reactivity through "domain-swaping"mutagenesis.
Thanks anonymous - this is a really nice paper!
Readers, the pubmed link is here:
http://www.ncbi.nlm.nih.gov/pubmed/27376689
Post a Comment