RVX-208, an Inducer of ApoA-I in Humans, Is a BET Bromodomain Antagonist

<div><p>Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes <i>in vitro</i>, and <i>in vivo</i> in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.</p></div

X-ray crystal structure of RVX-208 bound to BRD4[BD1].

<p>A. Surface rendering model showing disposition of RVX-208 in hydrophobic pocket. B. A closer view of the BRD4[BD1] ligand binding site. The electron density (sigmaA-weighted, 1σ contour level, 1.24 Å resolution) corresponding to the compound is shown in blue. For clarity, only interacting residues and some water molecules are shown with a ribbon diagram of the protein main chain course.</p

Schematic of domain structure of the human BET proteins.

<p>Bromdomains are designated by BD1 and BD2 and the Extraterminal domains by ET.</p

Stimulation of ApoA-I and inhibition of IL-6 production by RVX-208 and JQ-1.

<p>A. Dose dependent induction of ApoA-I in Huh7 cells by RVX-208 and JQ-1 at 48 h, B. Selective dose dependent inhibition of IL-6 but not TNFα mRNA in U937 cells stimulated with LPS for 4 h.</p

Binding of RVX-208 to tandem bromodomains [BD1 BD2].

<p>Binding of RVX-208 to tandem bromodomains [BD1 BD2].</p

Thermodynamics parameters for binding of RVX-208 to purified BET bromodomains obtained from isothermal titration calorimetry.

<p>Thermodynamics parameters for binding of RVX-208 to purified BET bromodomains obtained from isothermal titration calorimetry.</p

X-ray data reduction statistics and crystal parameters.

<p>^a Numbers in the parentheses are for the highest resolution shell of 1.28-1.24 Å for BRD4[BD1]/RVX-208) and 1.12-1.08 Å for BRD2[BD2]/RVX-208.</p

Selective binding of RVX-208 to bromodomains of the BET family.

<p>A. Competition of RVX-208 for binding to BRD4 bromodomains 1 and 2 individually or in tandem as measured in a competition FRET assay with acetylated histone 4 peptide. B. Same as A except for JQ-1. C. TR-FRET IC₅₀ for binding of 23 RVX-208 analogues to Brd4 [BD1BD2] is plotted against EC170 for induction of ApoA-I mRNA in Huh7 cells.</p

Binding of RVX-208 to individual bromodomains.

<p>Binding of RVX-208 to individual bromodomains.</p