Development of cell-active BRD4-D1 selective inhibitors to decode the role of BET proteins in LPS-mediated liver inflammation

Abstract

The endogenously expressed BET proteins (BRD2, BRD3, BRD4) are upstream clinical targets for anti-inflammatory treatments, where inhibition of the tandem bromodomains (D1 and D2) have proven efficacious in vitro and in vivo towards NF-κB-mediated inflammation. Despite their efficacy, dose-limiting toxicities associated with BET inhibition have limited clinical progression. One strategy to circumvent these dose-limiting toxicities has included domain- or protein-selective inhibition of the BET bromodomains. Based on previously reported 1,2,4-substituted imidazole scaffolds, we characterize and report on next-generation BRD4-D1 selective inhibitors, 39 and 41. Compound 39 is both highly potent and selective towards BRD4-D1 (K_i = 2.9 ± 1.0 nM, >1700-fold over BRD2-D1 via fluorescence anisotropy) over other BET bromodomains in addition to being cell-active at nanomolar concentrations. We also characterized 39's solubility and cellular activity in addition to its off-target hERG liability (a common cardiovascular risk for drug candidates). An acetylated analogue, 41, had an 80-fold reduced hERG affinity compared to previous BRD4-D1 selective compounds. In the context of liver inflammation, we screened 39 against an LPS-mediated cellular model of liver inflammation. Upon treatment with 39, pro-inflammatory chemokines CXCL1 and CCL2 transcripts were significantly downregulated compared to the control; however, BRD4-D1 selective inhibition remained insufficient to reproduce the anti-inflammatory activity of pan-BET treatment. On a mechanistic level, these data highlight that more than one bromodomain within the BET family may be contributing to CXCL1 and CCL2 expression, where multi-domain inhibition or other therapeutic modalities may be needed in these contexts to achieve sufficient anti-inflammatory effects.

Keywords: Anti-inflammatory; Bromodomain; Cell-active; Epigenetics; Liver disease.

Figure 1.. Previous Developments Towards BRD4-D1 Selective Inhibitors.

(A) Structures of previous compounds reported for inhibiting BET-bromodomains with differential selectivity profiles. (B) Structure of previously reported BRD4-D1 selective inhibitor, 30, with interactions with BRD4-D1 highlighted. IC₅₀ reported by fluorescence anisotropy (FA).[25] (C) Compound 30 co-crystalized with BRD4-D1 to highlight key interactions of structured-water displacement (red), phenyl ether (magenta), and the solvent exposed region (blue). Water-mediated hydrogen bond with D144 shown with dashed yellow lines. PDB: 7R9C.

Figure 2.. Structure-Activity Relationships of Tri-Substituted Imidazole Scaffold for BRD4-D1.

All IC₅₀ values were collected in experimental triplicates as technical replicates in a competitive FA assay for BRD4-D1, reported as mean ± s.d. IBET-151 data was collected as 12 experimental replicates of technical triplicates. Compounds in red represent IC₅₀ values more potent than 1. (A) Substitution in the water-displacement region (red) of 1. (B) Substitutions to the solvent-exposed region (blue) of 1. (C) Modifications to the piperidine-motif (green) of 1 to access acidic dyad. (D) Modifications to (S)-pyrrolidine (purple) to access interactions with BRD4-D1. (E) Screen of phenolic ethers (pink) in 1. (F) Combinative structure activity relationships and analogues (black).

Figure 3.. Co-Crystal Structure of 9 with BRD4-D1.

(A) Structured-water displacement region and key acetyl-lysine mimic when bound to 9 (PDB: 9P34). (B) Solvent-exposed region of 9 with a two-water bridge to D144, mimicking the interactions seen by previously reported BRD4-D1 selective inhibitors. Hydrogen bond shown by yellow dashed lines, distance measurement shown in Å.

Figure 4.. Selectivity Analysis of 39 and 41 Towards BET Bromodomains.

(A) Differential Scanning Fluorimetry of BRD4 (black), BRD3 (red), and BRD2 (green) bromodomains with BRD4-D1 selective inhibitors. (B) AlphaScreen selectivity of BRD4 (black), BRD3 (red), and BRD2 (blue) bromodomains of 39 from ReactionBiology. Refer to Supplemental Tables S6 and S7 for additional information.

Figure 5.. Cellular Target Engagement of BRD4-D1 Selective Inhibitors.

(A) 3-point scan of BRD4-D1 selective inhibitors against NLuc-BRD4-D1 in HEK293T. Compounds were incubated for 2 h at 37 °C. Error bars represent s.d. as biological triplicates. Blue: Acyl-motif containing analogues White: cyclopentyl analogues Orange: cyclobutyl analogues Green: Amide-containing analogues (B) Binding isotherms for IBET-151 (black), 39 (red), and 41 (blue). Error bars represent s.d. as biological triplicate of technical triplicates for 3-fold dilutions. (C) Table of EC₅₀ and K_i,app values for competition experiment shown in (B). EC₅₀ and K_i,app are shown as mean ± s.e.m. from biological triplicates from technical replicates. ^aValues for 1 and 2 are from previous reports.[21]

Figure 6.. CXCL1 and CCL2 Expression Levels in LPS-Stimulated TSECs.

qPCR expression CXCL1 (left) and CCL2 (right) in LPS-stimulated TSEC cells dosed with 1.2 μM of inhibitor. Data shown as mean ±s.d. of experimental duplicates of technical triplicates. * = p<0.05 ** = p<0.01 *** = p<0.0001 n.s. = non-significant.

Figure 7.. Structure-Activity Relationships to hERG Inhibitory Activity for BRD4-D1 Selective Inhibitors.

Structures of compounds screened against hERG activity in patch-clamp assay by Pharmaron. Analogues that probed hydrophobicity (orange) and inductive and acylation effects (blue) were screened, along with combinatory scaffolds (i.e., 41).