In vitro activity: OTX015 inhibits the binding of BRD2, BRD3, and BRD4 to AcH4 with IC50 ranging from 92 to 112 nM, and inhibits the growth of a variety of human cancer cell lines with GI50 ranging from 60 to 200 nM. OTX015 results in rapid down-regulation of c-MYC expression, and show the synergistic anti-proliferative effects in combination with ALK inhibitors in ALKpos ALCL cell lines.

Kinase Assay: To assess binding of OTX015 to BRD2, BRD3, and BRD4, BRD-expressing CHO cell lysate (from CHO cells transfected with expression plasmids for Flag-tagged BRD2, BRD3, or BRD4 or vector alone), europium-conjugated anti-Flag antibody, XL-665-conjugated streptavidin, and biotinylated OTX015 were incubated at room temperature for 0.2 to 2h. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and EC50 for binding was calculated by nonlinear regression using PRISM version 5.02. Using a similar system, the effect of OTX015 on binding of BRD2, BRD3, and BRD4 to acetylated histone H4 (AcH4) was evaluated by incubating biotin-conjugated -AcH4, BRD-expressing CHO cell lysate, europium-conjugated anti-Flag antibody, and XL-665-conjugated streptavidin. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and percent binding was calculated by defining the value of the sample without biotin conjugate dAcH4 as 0% and the sample without OTX015 as 100%. The IC50 value was calculated by nonlinear regression using PRISM version 5.02.

Cell Assay: Effects of OTX015 on cancer cell proliferation are evaluated by incubating human tumor cells for 72 h with increasing concentrations of OTX015 and assessing proliferation using a tetrazolium salt (WST-8)-based colorimetric assay.

Oncotarget. 2015 Mar 20;6(8):5501-16; Mol Cancer Ther. 2013, 12, C244; Mol Cancer Ther. 2013, 12, A219.