RVX-297- a novel BD2 selective inhibitor of BET bromodomains.

Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. We demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains.

Combination of ZEN-3694 with CDK4/6 inhibitors reverses acquired resistance to CDK4/6 inhibitors in ER-positive breast cancer.

CDK4/6 inhibitors significantly prolong progression-free survival in patients with advanced hormone receptor-positive (HR+) HER2-negative breast cancer. Despite recent successes, patients acquire resistance, necessitating the development of additional novel therapeutic strategies. Bromodomain and extra-terminal domain (BET) proteins are key epigenetic regulators that interact with acetylated lysine (AcLys) residues of histones or transcription factors. BET proteins are directly involved in modulating estrogen receptor (ER) signaling and the cell cycle. Therefore, BET inhibitors can potentially offer new strategies in the treatment of advanced ER+ breast cancer. ZEN-3694 is an orally bioavailable small molecule BET inhibitor currently being evaluated in Phase 1/2 clinical trials (NCT03901469). To assess a potential combination strategy in a CDK4/6i resistant breast cancer population, we investigated the mechanism of action of ZEN-3694 combined with CDK4/6 inhibitors in the ER+ cell lines resistant to palbociclib or abemaciclib. Here, we describe that the combination of ZEN-3694 with CDK4/6i potently inhibits proliferation and induces apoptosis in CDK4/6i resistant cell lines. The resistance to both palbociclib and abemaciclib was associated with the strong upregulation of CDK6 and CCND1 protein levels, which was reversed by the ZEN-3694 treatment. Furthermore, RNAseq data and pathway analysis elucidated the combinatorial effects of ZEN-3694 with CDK4/6 inhibitors through significant downregulation of multiple pathways involved in cell cycle regulation, cellular growth, proliferation, apoptosis, inflammation, and cellular immune response. Our data indicate that ZEN-3694 has therapeutic potential in combination with CDK4/6 inhibitors in patients with advanced ER+ breast resistant to CDK4/6 inhibitors.

Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine.

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

RVX-208, a BET-inhibitor for treating atherosclerotic cardiovascular disease, raises ApoA-I/HDL and represses pathways that contribute to cardiovascular disease.

High density lipoproteins (HDL), through activity of the main protein component apolipoprotein A-I (ApoA-I), can reduce the risk of cardiovascular disease (CVD) by removing excess cholesterol from atherosclerotic plaque. In this study, we demonstrate that the bromodomain and extraterminal domain (BET) inhibitor RVX-208 increases ApoA-I gene transcription and protein production in human and primate primary hepatocytes. Accordingly, RVX-208 also significantly increases levels of ApoA-I, HDL-associated cholesterol, and HDL particle number in patients who received the compound in recently completed phase 2b trials SUSTAIN and ASSURE. Moreover, a post-hoc analysis showed lower instances of major adverse cardiac events in patients receiving RVX-208. To understand the effects of RVX-208 on biological processes underlying cardiovascular risk, we performed microarray analyses of human primary hepatocytes and whole blood treated ex vivo. Overall, data showed that RVX-208 raises ApoA-I/HDL and represses pro-inflammatory, pro-atherosclerotic and pro-thrombotic pathways that can contribute to CVD risk.

Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes.

Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208.