4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity

Abstract

The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein−protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure−activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 K d of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 and 26 are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.

A Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes

“…GSK778, a potent pan-D1 inhibitor, was reported by Gilan et al The pyrrolidinyl group interacts with a nonconserved Asp on D1s (His on D2s) via a water-bridged hydrogen bond (Figure A,B) . Similar interactions were found by our recently reported triazole-based inhibitors, including DW34 , which exhibit pan-D1 selectivity, with the exception of a high affinity interaction with BRD4 D2 (Figure A,C) . Meanwhile, pan-BD2 inhibitors have been developed, including RVX-208, ABBV-744, and GSK046, and are effective in different disease models.…”

“…Affinities reduced sharply when polar ether or hydroxyl groups were installed ( 16 , 17 , 18 ). These results indicated that the aryl groups on the imidazole scaffold form significant hydrophobic interactions with the WPF shelf, which was not observed in our triazole-based inhibitor series …”

“…Here, several phenyl ethers with alkyl substituents of varying size and hydrophobicity were substituted on the pyrimidine ring ( 7 – 18 , Tables and ). Surprisingly, more dramatic changes were observed on the imidazole than on our previously reported triazole scaffold . When the exocyclic NH of 5 was replaced by an ether bond ( 7 , IC 50 = 0.19 μM), the affinity against BRD4 D1 was reduced by 2.4-fold.…”

“…Previously we installed an ethylamino or N , N -dimethyl-ethylamino group on the solvent exposed piperidyl group of the imidazole scaffold to target a D1-conserved Asp144 on BRD4 D1 for higher affinity and D1/D2 selectivity . However, on the basis of the cocrystal structure, these amino groups were unable to engage the conserved water molecule directly to target Asp144 similar to GSK778 and DW34 . ,, Analysis of our prior cocrystal structures supported a pyrrolidinyl group for orienting the amino group to target the conserved water molecule binding to Asp144. The two enantiomers, 19 and 21 , lacking an ethylamino group, had 5.8- and 10-fold weaker affinity relative to 7 (Table ).…”

“…We next evaluated changes to substituents on the pyrimidine ring of our imidazole scaffold, which interacts with a hydrophobic portion of BRD4 D1 called the WPF shelf (Figure C). Previously, we reported a strategy to improve BRD4 D1 affinity, and D1 vs D2 selectivity, by using different aryl groups targeting the WPF shelf on a related scaffold . Here, several phenyl ethers with alkyl substituents of varying size and hydrophobicity were substituted on the pyrimidine ring ( 7 – 18 , Tables and ).…”