Olesya A. Kharenko

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

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 in vitro, and in vivo 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.

Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design.

Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low μM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).

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.

Novel approaches to targeting BRD4

Inhibition of bromo and extra-terminal (BET) bromodomains, including BRD4, has emerged as a new exciting epigenetic target for oncology, in particular. Recently, novel alternatives to the traditional use of reversible small molecules have emerged, including proteolytic targeting BET agents and irreversible binding inhibitors. These alternatives to reversible inhibitors may offer some advantage and can be used as tools to further decipher the underlying biology. Supportive pre-clinical data have these novel approaches bound for clinical development in the near future.

Abstract LB-207: Preclinical characterization of ZEN-3694, a novel BET bromodomain inhibitor entering phase I studies for metastatic castration-resistant prostate cancer (mCRPC)

Metastatic castration resistant prostate cancer (mCRPC) is a major unmet medical need due to its widespread occurrence and incurable status. Current standard of care for advanced prostate cancer is androgen-deprivation therapy (ADT), and upon failure, patients are administered secondary ADT with androgen receptor (AR) antagonists such as enzalutamide and abiraterone. While most patients display an initial response to these agents, eventually all become resistant via various mechanisms that often result in constitutive AR signaling including mutations of the AR, and the generation of AR splice variants that bypass the ligand binding domain. Other mechanisms of resistance to AR antagonists include up-regulation of the glucocorticoid receptor (GR), and partial to complete loss of AR signaling through neuroendocrine differentiation. Recent evidence suggests that BET bromodomain inhibitors (BETi) could be efficacious in AR-signaling positive or negative mCRPC that are resistant to current therapies. ZEN-3694 is an orally bioavailable, potent BETi that selectively binds to both bromodomains of the BET proteins. In vitro, ZEN-3694 has demonstrated strong activity against several prostate cancer cell lines with submicromolar potency, including AR positive and AR negative, neuroendocrine, and enzalutamide resistant cell lines. In VCaP AR-positive prostate cancer cells, ZEN-3694 inhibited proliferation synergistically with enzalutamide, resulting in potent up-regulation of the CDKN1C/KIP2 tumor suppressor gene. In 22Rv1 cells displaying constitutive AR signaling through the AR-V7 splice variant, ZEN-3694 inhibited AR signaling, and in an in vitro LNCaP model of acquired resistance to enzalutamide characterized by GR up-regulation, ZEN-3694 decreased levels of GR in a dose-dependent manner. Furthermore, in the PC3 AR-null cell line, the expression of a subset of NF-KB-dependent genes reported to be involved in mCRPC bone metastasis was found to be inhibited by ZEN-3694. In vivo, using multiple prostate cancer cell line xenografts such as 22Rv1, and VCaP, ZEN-3694 showed efficacy in inhibiting tumor progression at well-tolerated doses, and modulating target gene expression. ZEN-3694 also inhibited progression of a patient-derived xenograft (PDX) LuCaP 35CR that is resistant to enzalutamide. In summary, our results indicate that ZEN-3694 demonstrates potent activity in advanced metastatic prostate cancer targeting multiple mechanisms of enzalutamide resistance in CRPC, including AR-V7 signaling and GR up-regulation in different preclinical models. This together supports the clinical development of ZEN-3694 as a single agent, and in combination with enzalutamide in mCRPC patients that have failed first line ADT. We are implementing a robust translational medicine program in the phase 1 study to measure target engagement and explore mechanisms of enzalutamide resistance and sensitivity to ZEN-3694 in patients. Citation Format: Sarah Attwell, Ravi Jahagirdar, Karen Norek, Cyrus Calosing, Laura Tsujikawa, Olesya A. Kharenko, Reena G. Patel, Emily M. Gesner, Eva Corey, Holly M. Nguyen, Sanjay Lakhotia, Henrik C. Hansen, Eric Campeau. Preclinical characterization of ZEN-3694, a novel BET bromodomain inhibitor entering phase I studies for metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-207.

Abstract C86: The clinical candidate ZEN-3694, a novel BET bromodomain inhibitor, is efficacious in the treatment of a variety of solid tumor and hematological malignancies, alone or in combination with several standard of care and targeted therapies

ZEN-3694 is an orally bioavailable small molecule discovered and developed from a BET bromodomain inhibitor discovery platform. In vitro, ZEN-3694 selectively binds to both bromodomains of the BET proteins, inhibiting the interaction of acetylated histone peptide with IC50 values in low nM range. ZEN-3694 inhibits proliferation of MV4-11 AML cells with an IC50 of 0.2 uM, and inhibits MYC mRNA expression with an IC50 of 0.16 uM. ZEN-3694 has also demonstrated strong activity against many solid tumor and hematological cell lines with sub-uM IC50 values. In vitro synergy with Standard of Care (SOC) agents has been shown in a wide variety of malignancies including Breast, Prostate, Lung, Melanoma, AML, and DLBCL. Xenograft studies conducted with ZEN-3694 in AML, prostate and breast cancer models have demonstrated that it is efficacious at well-tolerated doses, modulating target gene expression and halting tumor growth in a dose-dependent manner. In the AR positive VCAP prostate cancer cell line, ZEN-3694 inhibits proliferation synergistically with the AR antagonists enzalutamide and ARN-509. In an in vitro enzalutamide resistance model characterized by the up-regulation of the glucocorticoid receptor (GR), GR expression was inhibited by ZEN-3694 in a dose-dependent manner. Sensitivity to ZEN-3694 was unaltered, suggesting that it could be a valid therapeutic approach in patients developing resistance to AR antagonists through GR induction. Robust PD modulation has been observed across multiple matrices for ZEN-3694 and will be explored further in the clinic. Promising target validation data, excellent pharmacological properties, and robust activity of ZEN-3694 across a variety of hematological malignancy and solid tumor settings support the clinical development of ZEN-3694 in various oncologic indications. Citation Format: Sarah Attwell, Eric Campeau, Ravi Jahagirdar, Olesya Kharenko, Karen Norek, Laura Tsujikawa, Cyrus Calosing, Reena Patel, Emily Gesner, Sanjay Lakhotia, Henrik Hansen. The clinical candidate ZEN-3694, a novel BET bromodomain inhibitor, is efficacious in the treatment of a variety of solid tumor and hematological malignancies, alone or in combination with several standard of care and targeted therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C86.

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.