Bromodomain inhibitor i-BET858 triggers a unique transcriptional response coupled to enhanced DNA damage, cell cycle arrest and apoptosis in high-grade ovarian carcinoma cells.

BACKGROUND: Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. RESULTS: i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a 'core' network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. CONCLUSIONS: Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

Discovery of biaryl inhibitors of H+/K+ ATPase.

We report the identification of a novel biaryl template for H(+)/K(+) ATPase inhibition. Evaluation of critical SAR features within the biaryl imidazole framework and the use of pharmacophore modelling against known imidazopyridine and azaindole templates suggested that the geometry of the molecule is key to achieving activity. Herein we present our work optimising the potency of the molecule through modifications and substitutions to each of the ring systems. In particular sub-micromolar potency is achieved with (4b) presumably through a proposed intramolecular hydrogen bond that ensures the required imidazole basic centre is appropriately located.

Orally active C-6 heteroaryl- and heterocyclyl-substituted imidazo[1,2-a]pyridine acid pump antagonists (APAs).

Acid pump antagonists (APAs) such as the imidazo[1,2-a]pyridine AZD-0865 2 have proven efficacious at low oral doses in acid related gastric disorders. Herein we describe some of the broader SAR in this class of molecule and detail the discovery of an imidazo[1,2-a]pyridine 15 which has excellent efficacy in animal models of gastric acid secretion following oral administration, as well as a good overall developability profile. The discovery strategy focuses on use of heteroaryl and heterocyclic substituents at the C-6 position and optimization of developability characteristics through modulation of global physico-chemical properties.