Kinase experiment:

Briefly, 200 μg of extracted cellular protein from HeLaS3 cells in 200 μL of 70 mM HEPES buffer (with 1 mM dithiothreitol (DTT), 5 mM EDTA, pH 7.8) is incubated at 37℃ with a defined concentration of Lomeguatrib (added as a DMSO solution). After 30 min an excess of [3H]-methylated DNA (120 000 cpm) is added, and the incubation is continued for an additional 90 min. The reaction is stopped by the addition of 400 μL TCA (13%), and the DNA is hydrolyzed by heating the reaction mixture for 30 min at 98℃. The precipitated protein is washed three times with 400-μL portions of 5% TCA, solubilized in 0.1 N NaOH, and analyzed by liquid scintillation counting using the cocktail Rotiszint eco plus and a TRI-CARB. Enzyme activity is expressed as fmol of [3H]methyl transferred to TCA-insoluble protein material per mg of total cellular protein. Percent inhibition is calculated relative to untreated control samples. Each assay is repeated three times, and IC50 values are determined graphically from plots of percent inhibition vs inhibitor concentration[1].

Cell experiment:

To determine toxicity, the MTT growth inhibition assay is employed. Cells (1000 per well) are plated into a 96-well plate and following a 24 h attachment period, Lomeguatrib is added to the cells. After 2 h incubation with Lomeguatrib (10 μM) at 37℃, 5% CO2, increasing doses of temozolomide or vehicle are added and the cells are incubated for a further 4-5 days. At the end of the exposure period, 150 μg MTT is added to each well and plates are incubated for 3 h at 37℃, 5% CO2. The media are removed and the formazan crystals formed in the viable cells are solubilised in 200 μL DMSO. The absorbances at 540 and 690 nm are determined using a ELISA plate reader and growth inhibition calculated as a percentage of the A540-A690 of untreated wells[2].

Animal experiment:

Mice[2]To assess the ability of Lomeguatrib to sensitise human breast tumour xenografts to the tumour growth inhibitory effects of temozolomide, groups of at least six nude mice are treated as follows: the vehicle control group are given corn oil then 20% DMSO in PBS; the temozolomide only group are given corn oil then temozolomide (100 mg/kg/day); the Lomeguatrib only group are given Lomeguatrib (20 mg/kg/day) then DMSO in PBS, and the Lomeguatrib plus temozolomide group are given Lomeguatrib (20 mg/kg/day) then temozolomide (100 mg/kg/day). Drugs or vehicles are administered i.p. once daily for 5 days with a separation of 1 h. Up to 10 and at least six animals are assigned to each group, and mean tumour volume is standardised across the groups at the start of the experiment: thus the control, Lomeguatrib, temozolomide and Lomeguatrib plus temozolomide groups had mean tumour volumes of 29.8±7.6 (range 19.0-38.7), 33.2±14.7 (range 16.5-58.7), 35.1±10.9 (range 20.9-52.4) and 30.3±10.0 (range 20.7-44.5) mm3, respectively[2].

Lomeguatrib is an O6-methylguanine-DNA-methyl-transferase (MGMT) inhibitor [1], with an IC50 value of about 6 nM to inactivate MGMT in MCF-7 cells, effectively [2].

MGMT activity is closely related to MTIC (a metabolite of dacarbazine)-mediated DNA damage [1].

Lomeguatrib sensitized MGMT-activity-bearing A375P cells to temozolomide (TMZ), but it failed to affect the effect of dacarbazine (DTIC). In other mutBRAF cells and several mutNRAS cell lines such as WM1361, similar results were obtained [1].

With one exception, patients treated with lomeguatrib showed no active or very low MGMT in PBMCs. Lomeguatrib at a dose of 20 mg resulted in 16.7 fmol/μg DNA active MGMT in a CNS-tumor-bearing patient. This patient showed a percentage of 25% for inactive tumor MGMT. This percentage was lower than that in the other two CNS patients with lomeguatrib at the same dose. Different tumor types showed remarkable differences in total tumor MGMT. Prostate cancers had the highest (554 ± 404 fmol/mg protein), CNS tumors had the lowest (89.9 ± 44.5 fmol/mg protein), and colorectal tumors had intermediate levels of total protein (244 ± 181 fmol/mg protein). In the colorectal cancer, the primary CNS tumor, and the prostate cancer of patients, increasing lomeguatrib doses resulted in increasing inactive MGMT proportions [3].

References:
[1]. Imanol Arozarena, Ibai Goicoechea, Oihane Erice, et al. Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells. Molecular Cancer, 2014, 13:154.
[2]. M Clemons, J Kelly, AJ Watson, et al. O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts. British Journal of Cancer, 2005, 93:1152-1156.
[3]. Amanda J. Watson, Ami Sabharwal, Mary Thorncroft, et al. Tumor O6-methylguanine-DNA Methyltransferase Inactivation by Oral Lomeguatrib. Clinical Cancer Research, 2010, 16(2):743-9.