Kinase experiment:

Enzyme assays are done in fluorescent polarization (FP) format. Human class I PI3Ks and PI3K-α mutants (E545K and H1047R) are produced in Sf9. 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% β-mercaptoethanol] and stop/detection buffer [100 mM HEPES (pH 7.5), 4 mM EDTA, 0.05% CHAPS]. FP reaction is run for 30 min at room temperature in 20 μL of reaction buffer containing 20 μM phosphatidylinositol 4,5-bisphosphate (PIP2), 25 μM ATP, and

Cell experiment:

MDA-MB-361, MDA-MB-468, T47D, MCF7, BT474, HT29, HCT116, DLD1, U87MG, H157, NCI-H460, A549, NCI-H1975, NCI-H1650, NCI-H2170, KB, 786-0, A498, MIA-PaCa-2, and PC3 cell lines are propagated at 37℃ in 5% CO2 incubators in supplier-recommended growth medium. Cell growth inhibition is determined using the CellTiter 96 AQueous proliferation assay. Data are collected after 72 h using a Wallac Victor2 V 1420 multilabel HTS counter. FOXO-GFP translocation in U2OS cells is quantified after 60-min PKI-402 exposure using a Cellomics ArrayScan VTI Reader[1].

Animal experiment:

Mice[1]PKI-402 or vehicle is administered by i.v. route. Nude mice bearing MDA-MB-361 tumors are used. Tumor weight is calculated. Pharmacodynamic (biomarker) measurements are done on tumor-bearing female nude mice administered PKI-402. Tumor or normal tissue samples are collected from euthanized animals, homogenized, washed twice with cold (4℃) PBS, and then treated with cell lysis buffer[1].

• Heliyon (2022): e09068.

PKI-402 is a selective, equipotent and ATP-competitive class I PI3K inhibitor (IC50= 1 nM, 7 nM, 16 nM and 14 nM for PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, respectively.)

PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase) is a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. It plays a key role in PI3K/Akt/mTOR pathway.

In multiple human cancer cell lines (e.g. breast, brain, pancreas and NSCL), PKI-402 inhibited growth of cancer cells, and attenuated phosphorylation of effector of PI3K and mTOR. In MDA-MB-361, 30 nM PKI-402 caused cleaved of the apoptosis marker—PARP.

In MDA-MB-361 mouse tumor xenograft models, administration of 100 mg/kg of PKI-402 daily for 5 days decreased the initial tumor volume from 260 mm³to 129 mm³and inhibited tumor regrowth for 70 days, single dose of PKI-402 blocked phosphorylation of Akt and led to cleaved PARP.

Reference:

1. Mallon R, Hollander I, Feldberg L et al. Antitumor efficacy profile of PKI-402, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor. Mol Cancer Ther. 2010 Apr;9(4):976-84.