Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit

Abstract

The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine competitive and cell active inhibitor PFI-3 that binds to certain Family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for Family VIII was achieved through a novel bromodomain binding mode of a phenolic head group that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail and additional analogues with differing Family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.

Chemical probes targeting epigenetic proteins: Applications beyond oncology

“…The effect could be shown to be at least in part persistent as demonstrated by washout experiments 194 . Contrary, differentiation of myoblast to myotubes was inhibited by PFI-3, as was differentiation of adipocytes with severely decreased lipid accumulation in the adipocytes 196 . Also, BRD4 plays a role in early embryogenesis.…”

“…A highly specific BRD antagonist, PFI-3, has been developed and also shown to displace ectopically expressed SMARCA2 and SMARCA4 from chromatin 59,194 . However, no anti-proliferative effect could be observed in cell lines from several tumor types tested indicating that the ATPase domain may be more relevant for tumorigenesis, 194,195 nor was an effect seen in a series of primary human cells assaying inflammatory cytokine release 196 . However, studies using PFI-3 revealed the crucial role of the bromodomain in several differentiation processes 194,196 .…”

Large-scale analysis of water stability in bromodomain binding pockets with grand canonical Monte Carlo

“…The fragment led to the development of a nanomolar ligand, selective for the SMARCA and PB1 bromodomains (Family VIII). Gerstenberger et al 11 started from the same fragment and developed another potent chemical probe with a similar selectivity profile. At the same time, Sutherell et al 12 reported the discovery of another series of ligands, also targeting Family VIII and displacing all four waters from their pocket, based on a 2,3-dihydropyrrolo[1,2-a]quinazolin-5(1H)-one scaffold.…”

Chemical modulators for epigenome reader domains as emerging epigenetic therapies for cancer and inflammation

“…PFI-3 demonstrated excellent potency [PB1(5) K D = 54 nM, SMARCA2/SMARCA4 K D < 0.1 μM] and selectivity against >40 BRDs from other families. [41]…”