Apitolisib (GDC-0980, RG-7422, GNE-390)

PI3Kα (IC50 = 5 nM); PI3Kδ (IC50 = 7 nM); PI3Kγ (IC50 = 14 nM); PI3Kβ (IC50 = 27 nM); mTOR (Ki = 17 nM); TORC1; TORC2

Apitolisib (GDC-0980) is remarkably selective for a number of other members of the related PIKK family kinases, including C2alpha, C2beta, VPS34, PI4Kalpha, PI4Kbeta, and DNA-PK, with IC50 values of 1300 nM, 794 nM, 2000 nM, and 623 nM, respectively[1]. According to a recent study, apitolisib (GDC-0980) inhibits cell-cycle progression and induces apoptosis, with the greatest effectiveness against prostate, breast, and NSCLC cell lines (IC50 200 nM 29%, 500 nM 88%), and the least effectiveness against pancreatic and melanoma cell lines (IC50 200 nM 0%, 500 nM 33%)[2].

Apitolisib (GDC-0980) (1 mg/kg, p.o.) is undergoing phase I clinical trials for cancer and has shown significant efficacy in mouse xenografts. Apitolisib (GDC-0980) exhibits dose-proportional exposure from 5 mg/kg dosed in PEG to 50 mg/kg dosed in suspension in MCT, a finding that has been partially attributed to the drug's good solubility[1]. Clearance and PPB are also low. In 15 of the 20 xenograft models, apatolisib (GDC-0980) (5 mg/kg, p.o.) causes more than 50% TGI. The length of time that pAkt/tAkt has been knocked down is correlated with the variation in tumor response to Apitolisib (GDC-0980) therapy[2].

Enzymatic activity of the Class I PI3K isoforms is measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidyl inositide-3-phosphate product results in a decrease in fluorescence polarization signal as the labeled fluorophore is displaced from the GRP-1 protein binding site. Class I PI3K isoforms are expressed and purified as heterodimeric recombinant proteins. PI3K isoforms are assayed under initial rate conditions in the presence of 10 mM Tris (pH 7.5), 25 μM ATP, 9.75 μM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 2% (v/v) DMSO at the following concentrations for each isoform: PI3Kα,β at 60 ng/mL; PI3Kγ at 8 ng/mL; PI3Kδ at 45 ng/mL. After assay for 30 minutes at 25°C, reactions are terminated with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 μg/mL GRP-1 detector protein before reading fluorescence polarization on an Envision plate reader. IC50s are calculated from the fit of the dose−response curves to a 4-parameter equation.Human recombinant mTOR(1360−2549) is expressed and purified from insect cells and assayed using a Lanthascreen fluorescence resonance energy transfer format in which phosphorylation of recombinant green fluorescent protein (GFP)-4-EBP1 is detected using a terbium-labeled antibody to phospho-threonine 37/46 of 4-EBP1. Reactions are initiated with ATP and conducted in the presence of 50 mM Hepes (pH 7.5), 0.25 nM mTOR, 400 nM GFP-4E-BP1, 8 μM ATP, 0.01% (v/v) Tween 20, 10 mM MnCl2, 1 mM EGTA, 1 mM dithiothreitol, and 1% (v/v) DMSO. Assays are conducted under initial rate conditions at room temperature for 30 minutes before terminating the reaction and detecting product in the presence of 2 nM Tb-anti-p4E-BP1 antibody and 10 mM EDTA. Dose−response curves are fit to an equation for competitive tight-binding inhibition and apparent Ki' s are calculated using the determined Km for ATP of 6.1 μM.

Antiproliferative cellular assays are conducted using PC3 and MCF7.1 human tumor cell lines. MCF7.1 is an in vivo selected line and originally derived from the parental human MCF7 breast cancer cell line. Cell lines are cultured in RPMI supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, 10 mM HEPES, and 2 mM glutamine at 3°C under 5% CO2. MCF7.1 cells or PC3 cells are seeded in 384-well plates in media at 1000 cells/well or 3000 cells/well, respectively, and incubated overnight prior to the addition of GDC-0980 to a final DMSO concentration of 0.5% v/v. MCF7.1 cells and PC3 cells are incubated for 3 days and 4 days, respectively, prior to the addition of CellTiter-Glo reagen and reading of luminescence using an Analyst plate reader. For antiproliferative assays, a cytostatic agent such as aphidicolin and a cytotoxic agent such as staurosporine are included as controls. Dose−response curves are fit to a 4-parameter equation and relative IC50s are calculated using Assay Explorer software.

In Hank's Balanced Salt Solution, human prostate cancer PC3 cells are reconstituted, and 3 106 cells are implanted subcutaneously into the right hind flank of athymic nu/nu (nude) mice. Prior to starting treatment, tumors are watched until their mean tumor volume reaches 150–200 mm3. Athymic nu/nu (nude) mice with the right hind flank subcutaneously implanted with 5106 MCF7.1 cells that have been resuspended in a 1:1 mixture of Hank's Buffered Salt Solution and Matrigel Basement Membrane Matrix. Each naked mouse has a 0.36 mg/pellet of 17-estradiol (60-day release, no. SE-121) implanted beneath its dorsal shoulder blade before the cell inoculation. Tumors are watched after cell implantation until their mean tumor volume reaches 250–350 mm3 before dosing begins. 0.5% methylcellulose and 0.2% Tween-80 (MCT) are used to dissolve compound 2. Charles River Laboratories provides female nude (nu/nu) mice that are 20-30 g in weight, 6-8 weeks old, and in this condition. Depending on the xenograft model, tumor-bearing mice receive daily oral doses of 100 L of the test agent or vehicle (MCT) for 14–21 days.

[1]. Discovery of a potent, selective, and orally available class I phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer. J Med Chem, 2011, 54(21), 7579-7587.

[2]. GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models driven by the PI3K pathway. Mol Cancer Ther, 2011, 10(12), 2426-2436.

C23H30N8O3S

498.6011

498.22

C, 55.40; H, 6.06; N, 22.47; O, 9.63; S, 6.43

1032754-93-0

1032754-93-0

white solid powder

C[C@H](O)C(N1CCN(CC2=C(C)C3=NC(C4=CN=C(N)N=C4)=NC(N5CCOCC5)=C3S2)CC1)=O

YOVVNQKCSKSHKT-HNNXBMFYSA-N

InChI=1S/C23H30N8O3S/c1-14-17(13-29-3-5-31(6-4-29)22(33)15(2)32)35-19-18(14)27-20(16-11-25-23(24)26-12-16)28-21(19)30-7-9-34-10-8-30/h11-12,15,32H,3-10,13H2,1-2H3,(H2,24,25,26)/t15-/m0/s1

(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one.

GNE-390; GNE390; Apitolisib; GDC0980; GDC-0980; RG-7422; RG 7422; GNE 390; GDC 0980; RG7422

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~20 mg/mL (40.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 1.43 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.43 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.43 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose+0.2%Tween 80: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)