Biacore studies

Competition assays were performed on a Biacore S51. A Series S Sensor chip CM5 was derivatized for immobilization of a PentaHis antibody for capture of the His-tagged p53. The level of capture was ~ 200 response units (1 response unit corresponds to 1 pg of protein per mm2). The concentration of MDM2 protein was kept constant at 300 nM. Nutlin-3a was dissolved in DMSO at 10 mM and further diluted to make a concentration series of Nutlin-3a in each MDM2 test sample. The assays were run at 25°C in running buffer (10 mM Hepes, 0.15 M NaCl, 2% DMSO). MDM2-p53 binding in the presence of Nutlin-3a was calculated as a percentage of binding in the absence of Nutlin-3a and IC50 was calculated using Microsoft Excel.

Cell lines

HCT116 and SW480 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

1, 2, 4, 8 and 16 μM; 8 hrs

Applications

Nutlin-3a increased the cellular levels of p53, MDM2 and p21Waf1/Cip1 in HCT116 cells whilst SW480 cells exposed to the same conditions showed high basal levels of p53 but no detectable MDM2 or p21.

Animal models

Nude mice bearing subcutaneous human cancer xenografts (SJSA-1)

Dosage form

200 mg/kg; p.o.; b.i.d., for 20 days

Applications

Nutlin-3a inhibited tumor growth by 90%. The mice did not lose significant weight and did not show any gross abnormalities upon necropsy at the end of the treatment.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 2004, 303(5659): 844-848.