Kinase experiment:

Cells are then lysed and sonicated in 50 mM Hepes pH 7.5, 150 mM NaCl, 20 mM EDTA, 1 mM PMSF, 10 μg/mL leupeptin, 2 μg/mL aprotinin and 0.2% NP-40. Equal amount of lysates are mixed in substrate buffer (50 mM Hepes, 100 mM NaCl, 1 mM EDTA, 10% sucrose, 0.5% CHAPS, 5 mM dithiothreitol) with Ac-DEVD-AMC substrate and caspase-3/7 substrate in a microtiter plate. Production of fluorigenic substrate is measured continuously at 37℃ in a spectrophotometer Ascent Fluoroskan and the caspase activity (expressed as U/mg of protein) is defined as the amount of enzyme cleaving 1 nmol of substrate/min.

Cell experiment:

Colon cancer cells are seeded at a density of 104 cells cm2 and incubated either in serum-enriched medium or in base medium (containing 0.1% bovine serum albumin, BSA) supplemented or not with various concentrations of rCXCL9, rCXCL10 and rCXCL11 for the indicated periods of time before being either trypsin-detached, collected and enumerated or re-fed with fresh medium for 3 days, harvested and enumerated. The morphology of the CRC cells is observed through an inverted optical microscope at ×20 magnification, and photographs are taken at day 7.

Animal experiment:

Local tumor growth and spontaneous metastasis are evaluated by injecting 3×105 viable tumor cells s.c. proximal to the right abdominal mammary gland of syngeneic female mice. Tumor diameters are measured by caliper twice weekly, and mice are euthanized on an individual basis when the s.c. tumor measured 18 mm in diameter or earlier if the mouse seemed moribund. The lungs are removed and weighed, and surface tumor colonies are quantified in a blinded fashion under a dissecting microscope. Experimental metastasis is evaluated by injecting 9×104 viable tumor cells i.v. into the lateral tail vein of syngeneic female mice. All mice are euthanized on day 21 posttransplantation or earlier if the mice seemed moribund. The lungs are removed and weighed, and surface tumor colonies are quantified in a blinded fashion under a dissecting microscope. A 50% hydroxypropyl-β-cyclodextrin solution is prepared; at 20%, this solution serves as the vehicle. AMG487 is added to the 50% solution, and it is incubated in a sonicating water bath for 2 hours with occasional vortexing. Distilled water is added to give the appropriate final concentration of AMG487 in 20% of hydroxypropyl-β-cyclodextrin.

AMG 487 is a potent and selectiveantagonist of chemokine (C-X-C motif) receptor 3 (CXCR3) with IC50 values of 8nM and 8.2nM for I-IP-10 and I-ITAC, respectively [1].

AMG 487 is an 8-azaquinazolinone, it can prevent the chemokines I-IP-10 and I-ITAC from binding to CXCR3. In the cellular assays, AMG 487 inhibits CXCR3-mediated cell migration with IC50 values of 8nM, 15nM and 36nM for I-IP-10, I-ITAC and MIG, respectively. It is also found to inhibit ITAC induced calcium mobilization with IC50 value of 5nM. In the cells, AMG 487 is converted into M1 (pyridyl N-oxide AMG 487) and M2 O-deethylated AMG 487) by CYP3A4 and CYP3A5. It is reported that the metabolite M2 can inhibit CYP3A in a competitive manner with Ki value of 0.75μM [1, 2].

References:
[1] Johnson M, Li AR, Liu J, Fu Z, Zhu L, Miao S, Wang X, Xu Q, Huang A, Marcus A, Xu F, Ebsworth K, Sablan E, Danao J, Kumer J, Dairaghi D, Lawrence C, Sullivan T, Tonn G, Schall T, Collins T, Medina J. Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3. Bioorg Med Chem Lett. 2007 Jun 15;17(12):3339-43.
[2] Tonn GR, Wong SG, Wong SC, Johnson MG, Ma J, Cho R, Floren LC, Kersey K, Berry K, Marcus AP, Wang X, Van Lengerich B, Medina JC, Pearson PG, Wong BK. An inhibitory metabolite leads to dose- and time-dependent pharmacokinetics of (R)-N-{1-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxy-phenyl)-acetamide (AMG 487) in human subjects after multiple dosing. Drug Metab Dispos. 2009 Mar;37(3):502-13.