In vitroactivity: LY3023414 shows high solubility across a wide pH range. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 causes G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In cell-based assays, LY3023414 inhibition of PI3K and mTOR is assessed in the PTEN-deficient U87 MG glioblastoma cell line. LY3023414 inhibits the phosphorylation of AKT at position T308 downstream of PI3K at an IC50 of 106 nM. Similarly, LY3023414 inhibits phosphorylation of AKT at position S473 (IC50 = 94.2 nM) by mTORC2 as well as phosphorylation of mTORC1 kinase targets p70S6K (position T389; IC50 =10.6 nM) and 4E-BP1 (positions T37/46; IC50 = 187 nM). The downstream phosphorylation of S6RP at positions pS240/244 (IC50 = 19.1 nM) by p70S6K was inhibited as well, indicating target inhibition along the entire PI3K/AKT/mTOR pathway by LY3023414.

Kinase Assay: The selectivity and inhibitory potential of LY3023414 are assessed against a panel of 192 kinases in PC-3 cell lysates using the KiNativ platform and a panel of 102 kinases as purified enzymes from Cerep. Together, the 2 kinase panels covered approximately 266 unique kinases. These kinases are tested with three concentrations of LY3023414 to measure inhibition and calculate approximate IC50 values. The IC50 of LY3023414 for PI3Kα is measured using 5 nM recombinant human PI3Kα, 0.01 mM ATP with a 1.76 mM Triton X 100/0.04 mM PIP2/0.2 mM PS mixed micelle as the lipid substrate in a scintillation proximity assay (SPA) with neomycin-linked beads. The IC50 of LY3023414 for PI3Kβ is measured using a mixed micelle SPA format with 0.04 mM ATP with a 0.27 mM Triton X 100/0.05 mM PIP2/0.04 mM PA mixed micelle as the lipid substrate. The IC50s of PI3Kδ and PI3Kγand of DNA-PK are measured. The IC50 for mTOR is measured.

Cell Assay: In biochemical testing against approximately 266 kinases, LY3023414 was found to potently and selectively inhibit class I PI3K isoforms, mTORC1/2, as well as DNA-PK at low concentrations. In addition, inhibition of PI3K/AKT/mTOR signaling by LY3023414 led to G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel.