Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

Abstract

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein–protein interaction partners.

Selective Inhibition of Bromodomain-Containing Protein 4 Reduces Myofibroblast Transdifferentiation and Pulmonary Fibrosis

“…Importantly, ZL0591 does not target the highly conserved BD’s central binding pocket and is therefore capable of specifically binding to BRD4 BD1 with ~100 nM affinity, while demonstrating 10-fold lower affinity for BRD4 BD2 and BRD3 ( Liu et al, 2020 ; Zhou et al, 2021 ). In addition, ZL0591 administration disrupts poly (I:C)-induced H3K122 acetylation, confirming that ZL0591 directly engages with its BRD4 target in vivo ( Liu et al, 2022 ).…”

“…Third, the nonselectivity of JQ1 precludes an understanding of which BET protein mediates fibrosis, along with the potential of inducing off-target effects. Our study advances the field through a rigorous therapeutic experimental design using a highly selective BRD4 inhibitor that engages the BRD4 BD1 in a novel site ( Liu et al, 2022 ), and lacks evidence of hematologic supression ( Liu et al, 2018 ).…”

“…However, since JQ1 indiscriminately blocks the high affinity binding pockets of BRD2, BRD3, as well as both BD1 and BD2 of BRD4, it could not be utilized for assessment of the role of different BET family members or BDs in the fibrotic process. For this reason, we previously developed an array of selective inhibitors and utilized ZL0591, a selective allosteric inhibitor that targets BRD4 BD1 with ~100 nM affinity, while demonstrating low affinity for BRD4 BD2, BRD2 and BRD3, to enable elucidation of the role of BRD4 BD1 in pulmonary fibrosis ( Liu et al, 2020 ; Liu et al, 2022 ). We found that administration of ZL0591 during the fibrogenic stage of bleomycin-induced injury in mice attenuates fibrosis, demonstrating that BRD4 acts through its BD1 and is the member of the BET family responsible for mediating these pro-fibrotic effects.…”

Targeting key proteins involved in transcriptional regulation for cancer therapy: Current strategies and future prospective

“…The latest studies have demonstrated that allosteric molecules display outstanding performance at modulating those key proteins in transcriptional regulation, such as HIF-2α, STAT3, SIRT6, KRAS, BRD4, EED, and SHP2 (Figure 16). 140,329,[362][363][364][365][366][367] However, despite considerable progress with allosteric drugs having been made, which have gained significant attention from global R&D institutions and companies, several challenges in the determination of allosteric drugs and the illustration of the mechanism outstrip those underwent in the discovery of orthostatic drugs. The first challenge is related to the access to allosteric sites in only specific protein conformations, therefore, it is hard to determine the allosteric sites by X-ray crystallography or NMR spectroscopy.…”

Inhibition of BET Family Proteins Suppresses African Swine Fever Virus Infection

“…Apabetalone (RVX-208; BET inhibitor, S7295), ARV-825 (BRD4 specific inhibitor, S8297), AZD-5153 6-hydroxy-2-naphthoic acid (BET/BRD4 inhibitor, S8344), CPI-203 (BET inhibitor, S7304), Molibresib (I-BET-762; BET inhibitor, S7189), (+)-JQ1 (BET inhibitor, S7110), INCB054329 (BET inhibitor, S8753), MS436 (BET inhibitor, S7305), Birabresib (OTX015; BET inhibitor, S7360), PLX51107 (a new BET inhibitor, S8739), and PFI-1 (PF-6405761; BRD2/BRD4 inhibitor, S1216) were purchased from Selleck.cn, and ZL0580 (a BRD4-specific inhibitor) was prepared as previously described ( 20 , 45 ). The structures and functions of these inhibitors are shown in Fig.…”