Kinase experiment:

CNX-1351 is tested in a panel of 10 lipid kinases. CNX-1351s tested in a 10-concentration IC50 curve with 3-fold serial dilution starting at 1 μM. Reactions are carried out at 10 μM ATP. An HTRF assay format is used for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ; ADP-GLO assay format is used for other kinases. The following substrates are used: for HTRF, phosphatidylinositol 4,5-bisphosphate; for SPHK1 and SPHK2, sphingosine; for other ADP-GLO enzymes, phosphatidylinositol. For general kinase selectivity, CNX-1351 is run in a kinase selectivity panel at using HotSpot technology and radioisotope-based P81 filtration. CNX-1351 is dissolved in pure DMSO to the final 1 μM test concentration. Substrates for the various kinases tested against CNX-1351 are prepared fresh daily in reaction buffer. Any required cofactors are then added to the substrate solution followed by kinase addition and preincubated for 30 min at room temperature. 33P-ATP (10 μM) is delivered into the reaction mixture to initiate the reaction, and reaction continued for 2 h at room temperature. The reaction is terminated, and any unreacted phosphate is washed away using 0.1% phosphoric acid prior to detection utilizing a proprietary technology. The study is performed in duplicate, and 10 μM staurosporine, a nonselective, ATP-competitive kinase inhibitor, is used as the positive control[1].

Cell experiment:

SKOV3 cells or MCF-7 cells are plated in SKOV3 proliferation assay medium (DMEM supplemented with 5-10% FBS and pen/strep) at a density of 5000 cells in 180 μL volume per well in Costar no. 3610 white 96-well clear flat-bottom plates and incubated overnight in a humidified 37℃ incubator. A standard curve ranging from 10 000 to 50 000 cells is set up in a separate plate and allowed to adhere to the plate for 4-6 h, at which time the plate is developed using Cell Titer-Glo. The next morning, 3-fold compound dilutions ranging from 10 000 to 40 nM are prepared in proliferation medium containing 1% DMSO. Then 20 μL of each dilution is added to the SKOV3 or MCF-7 cells plated the previous day, resulting in a dose-response curve from 1000 to 4 nM. The cells are incubated for 96 h and then developed with Cell Titer Glo.The cell numbers at the end of the assay are determined using the standard curve generated at the start of the assay. Growth inhibition is calculated using the following formulas, and GI50 values are determined[1].

Animal experiment:

Mice[1] Female nu/nu (n=3/group) are administered compound (GDC-0941 or CNX-1351) delivered ip at 100 mg/kg once daily for 5 consecutive days. After delivery of the last dose, spleens from treated animals are harvested at 1, 4, and 24 h time points. Spleens are immediately frozen in liquid nitrogen. Samples are stored at -80℃ until processing for homogenates. Homogenates are made by adding approximately 100 μL of spleen sample to a Precellys homogenizing tube containing 300 μL of cell extraction buffer plus Complete protease inhibitor and PhosStop phosphatase inhibitor and kept on ice. The sample is homogenized in a Precellys 24 homogenizer for 15 s followed by centrifugation at 16000g for 20 min at 4℃. The supernatant is moved to a new tube, and the protein concentration is determined by BCA Assay.

CNX-1351 is a potent and isoform-selective targeted covalent PI3Kα inhibitor with IC50 of 6.8 nM.

CNX-1351 is able to potently (EC50PIK3CA activating mutations, SKOV3 ovarian cancer cells (H1047R) and MCF-7 breast cancer cells (E545K), are treated with CNX-1351 and growth is monitored. Both PIK3CA-driven cell lines are growth inhibited by exposure to CNX-1351 for 96 h (GI50 of 78 and 55 nM, respectively)[1].

CNX-1351 inhibits p-AktSer473 in mouse spleens and bonds to PI3Kα in vivo. CNX-1351 is delivered into the intraperitoneal cavity of nude mice at 100 mg/kg once a day for 5 consecutive days (n=3 mice per group). Spleens are harvested from the mice at the indicated times after the last dose (1-24 h) and interrogated by immunoblot for P-AktSer473 or for PI3Kα occupancy. Inhibition of PI3K signaling is detected as a decrease in P-AktSer473 at 1 and 4 h after last dose[1].

References:
[1]. Nacht M, et al. Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα. J Med Chem. 2013 Feb 14;56(3):712-21.