Gedatolisib (PKI-587)

PI3K, MTOR

615, 73

MDA-361 and H1975 cells are injected subcutaneously into the nude mice.

PKI-587 is currently in Phase I clinical trials in patients with solid tumors.

<=30 mg/kg

0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM

In nude mice, PKI-587 treatment at 25 mg/kg iv leads to low plasma clearance (7 (mL/min)/kg), high volume of distribution (7.2 L/kg), and long half-life, (14.4 hours). In the MDA-361 xenograft model, PKI-587 produces potent antitumor efficacy with the minimum efficacious dose (MED) of 3 mg/kg against MDA-361 tumors and maximum tolerated single dose (MTD) of 30 mg/kg. While in the H1975 (non-small-cell lung carcinoma, mutant EGFR [L858R, T790M]) xenograft model, PKI-587 at 25 mg/kg for 7 weeks results in 90% survival of the group treated. [1]

PI3K and mTOR kinase assay, Enzyme assays are done in fluorescent polarization (FP) format, adapted from the Echelon K-1100 PI3K FP assay kit protocol. Human class I PI3Ks and PI3K-alpha mutants (E545K and H1047R) are produced in Sf9 or purchased from Upstate Biotech. GST-GRP1 (murine) is produced in Escherichia coli and isolated by GST-Sepharose. Assay buffers are reaction buffer [20 mM HEPES (pH 7.1), 2 mM MgCl2, 0.05% CHAPS, and 0.01% beta-mercaptoethanol] and stop/detection buffer [100 mM HEPES (pH 7.5), 4 mM EDTA, 0.05% CHAPS]. FP reaction is run for 30 minutes at room temperature in 20 uL of reaction buffer containing 20 uM phosphatidylinositol 4, 5-bisphosphate (PIP2), 25 uM ATP, and <4% DMSO. FP reaction is stopped with 20 uL of stop/detection buffer (10 nM probe and 40 nM GST-GRP), and after 2 hours, data are collected using an Envision plate reader. The routine assays with purified FLAG-TOR (FL and 3.5) are performed in 96-well plates as follows. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM beta-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 uM microcystin LR, and 100 ug/mL BSA). To each well, 12 uL of the diluted enzyme is mixed briefly with 0.5 uL test inhibitor or control vehicle dimethyl sulfoxide (DMSO). The kinase reaction is initiated by adding 12.5 uL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 uL containing 800 ng/mL FLAG-TOR, 100 uM ATP, and 1.25 uM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1–6 hours) at room temperature with gentle shaking and then terminated by adding 25 uL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA).

DMSO 2 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

1-(4-(4-(dimethylamino)piperidine-1-carbonyl)phenyl)-3-(4-(4, 6-dimorpholino-1, 3, 5-triazin-2-yl)phenyl)urea