The use of complementary biophysical approaches to assess fragment binding to the epigenetic target bromodomain containing protein 3 (BRD3)

J. Lane¹, J. Morgan¹, A. Scott¹, D. Allcock², C. Sleigh³, S. Pollack¹, H. Williams⁴, D. Ogg⁵, T. Howard⁵, J. Brown³, K. Chohan³, S. Paoletta³, M. Abbott⁵, S. Onions³. ¹Bioscience, Sygnature Discovery, Nottingham, United Kingdom, ²Covance Laboratories Ltd., Harrogate, United Kingdom, ³Sygnature Discovery, Nottingham, United Kingdom, ⁴Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom, ⁵Peak Proteins, Alderley Edge, United Kingdom.

Background and aims: Fragment-based approaches have been applied successfully to the discovery and optimisation of ligands for a wide range of targets. Fragment-based drug discovery (FBDD) is proving particularly valuable for bromodomain (BRD)-containing proteins, an emerging family of epigenetic regulators with potential as targets for cancer, inflammation and multiple sclerosis. These proteins are able to recognize and read the acetylated lysine (KAc) modifications on histones and are subsequently responsible for transducing and translating signals into various normal or abnormal phenotypes (1). There are several BRD isoforms and compounds selective for individual BRDs would be useful pharmacological tools, with the potential to be therapeutic leads as well.

Summary of work: Sygnature’s fragment library was screened by surface plasmon resonance (SPR) to generate an initial set of hits against BRD3. These hits were then confirmed orthogonally by ligand-observed nuclear magnetic resonance (NMR), using saturation transfer difference (STD) experiments. Additional characterisation was undertaken by isothermal titration calorimetry (ITC), microscale thermophoresis (MST) and protein X-ray crystallography.

Discussion: Initial SPR fragment screening of ∼1000 compounds resulted in 275 actives that were further interrogated for sensorgram shape (i.e. square wave), effects on baseline and potential for superstoichiometric binding, this narrowed the selection to 148 hits. By performing concentration response titrations to calculate K_D for each of these, it was found that 20 fragments had repeatable, saturating concentration-dependent responses. BRD3/BD1 crystals were soaked with 100 mM fragments and data collected at the Diamond Light Source (Oxford, UK). Of the nine fragments analysed, six were found to contain bound fragments. Several orthogonal techniques where then used to validate the fragment hits including saturation transfer difference-NMR, MST and ITC.

Conclusions: Several fragments were identified that bind at the acetyl-lysine binding site of the first bromodomain in BRD3. These fragments were shown to bind in a variety of different orthogonal techniques, validating their binding affinities. Further chemical elaboration of these fragments could lead to a BRD3-selective molecule that could be utilized as a probe for elucidating the individual roles of the members of the BET-family.

References:

(1) Filppakopoulos P et al. (2010). Nature 468: 1067-1073