Molecule

Description

PKI-402 is a potent dual PI3K/mTOR inhibitor targeting PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ and mTOR with IC50 of 2 nM, 7 nM, 16 nM, 14 nM, and 3 nM, respectively.

Targets

IC₅₀

In Vitro

Equivalent to the IC50 for wild-type PI3Kα, PKI-402 inhibits E545K and H1047R PI3Kα mutants with IC50 of 3 nM. In a panel of 236 human protein kinases, PKI-402 only displays inhibitory activity against C-Raf and B-Raf with IC50 of 7 μM, and displays little activity against all other kinases with IC50 of > 10 μM. PKI-402 inhibits the growth of human tumor cell lines with IC50 of 6-349 nM. Consistently, PKI-402 inhibits phosphorylation of PI3K and mTOR effector proteins, particularly phosphorylated Akt (p-Akt) at T308 and S473 with IC50 of <10 nm="" and=""><30 nm,="" respectively.="" pki-402="" inhibits="" both="" p70s6k="" and="" 4ebp1="" phosphorylation="" with="" ic50="" of=""><10 nm.="" pki-402="" inhibits="" akt="" phosphorylation="" of="" pras40="" at="" t246="" with="" ic50="" of=""><30 nm,="" and="" inhibits="" akt="" phosphorylation="" of="" enos="" at="" s1177="" and="" gsk3α/gsk3β="" at="" s9/s21="" with="" ic50="" of=""><10 nm.="" in="" mdamb-361,="" a="" breast="" tumor="" line="" with="" mutant="" pi3k-α="" (e545k)="" and="" elevated="" levels="" of="" her2="" receptor,="" pki-402="" treatment="" induces="" cleaved="" poly(adp-ribose)="" polymerase="" (parp),="" a="" marker="" for="" apoptosis.="" less="" than="" 10%="" of="" mdamb-361="" cells="" exposed="" to="" pki-402="" at="" 0.3="" μm="" (or="" higher)="" for="" 24="" hours="" remain="" viable.="">^[1]

In Vivo

Single dose of PKI-402 (100 mg/kg) suppresses Akt phosphorylation (at T308) and induces cleaved PARP in MDA-MB-361 tumors. In normal tissue (heart and lung), PKI-402 (100 mg/kg) has minimal effect on p-Akt, with no detectable cleaved PARP. Consistently, PKI-402 at 100 mg/kg (daily for 5 days, one round) reduces initial tumor volume of 260 mm3 to 129 mm3 and prevents tumor regrowth for 70 days in MDA-MB-361. PKI-402 significantly inhibits the growth of A549 tumors in nude mice at 25 mg/kg and 50 mg/kg. PKI-402 at 100 mg/kg (daily for 5 days, one round) causes significant (P < 0.01)="" reduction="" in="" tumor="" growth="" of="" u87mg.="">^[1]

Clinical Trials

Features

Description

When HCT116 cells are exposed to 0.1 μM PD0325901 and PKI-402 (from 0.03 μM to 3.0 μM) for 24 hours, a substantial increase in cleaved PARP is observed. Consistently, PKI-402, or PD0325901, alone weakly induces caspase-3/7 activity at the highest compound concentration (3.0 μM). In contrast, PD0325901 (0.1 μM) combined with PKI-402 increases caspase-3/7 activity at PKI-402 concentrations from 0.03 μM to 3.0 μM (5-fold increase at 3.0 μM). ^[1]

Fluorescence polarization format assay

Enzyme assays are done in fluorescent polarization (FP) format, adapted from the Echelon K-1100 PI3K FP assay kit protocol. Assay reaction buffer is 20 mM Hepes, pH 7.5, 2 mM MgCl₂, 0.05% CHAPS, and 0.01% βME. Assay STOP/detection buffer is 100 mM Hepes, pH 7.5, 4 mM EDTA, 0.05% CHAPS. FP assays are run in Nunc 384-well black polypropylene fluor plates. The FP reaction is run in 20 μL of reaction buffer containing 20 μM PIP2, 25 μM ATP, and >4% DMSO. The reaction is run for 30 minutes at room temp. The reaction is stopped with 20 μL of STOP/detection buffer containing 10 nM probe and 40 nM GST-GRP. Assay plates are incubated for 2 hours, and fluorescence polarization is measured in a Perkin-Elmer Envision plate reader with TAMRA-FP filters.

Cell Lines

MDA-MB-361, MDA-MB-468, T47D, MCF7, BT474, HT29, HCT116, DLD1, U87MG, H157, NCI-H460, A549, NCI-H1975, NCI-H1650, NCIH2170, KB, 786-0, A498, MIA-PaCa-2, and PC3

Concentrations

Dissolved in DMSO, final concentrations ~3 μM

Incubation Time

72 hours

Methods

Cell growth inhibition is determined using the CellTiter 96 Aqueous nonradioactive cell proliferation assay. This homogeneous colorimetric method determined the number of viable cells in proliferation assays. The assay is carried out in 96-well format, with cell number numbers per well being adjusted on the basis of growth characteristics of the various cell lines used. Assay end point data are quantitated after 72 hours of PKI-402 exposure using a Victor2 V (Wallac) model 1420 multilabel HTS counter.

Animal Models

Female nude mice injected subcutaneously with MDA-361, U87MG, or A549 cells

Formulation

Dissolved in 0.5% methylcellulose and 0.4% polysorbate 80 (Tween-80)

Doses

~100 mg/kg/day

Administration

Administered by i.v. route