Biochemical binding affinity-HTRF assay

The p53-MDM2 HTRF assay was performed in buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT, 0.02 or 0.2 mg/mL BSA. Small-molecule inhibitors were stored in aliquots as 10 mM stock solutions in DMSO at 4℃ in 96-deep-well plates. It was thawed and mixed immediately prior to testing. The compound was incubated with GST-MDM2 and a biotinylated p53 peptide for one hr at 37 ℃. Phycolink goat anti-GST (Type 1) allophycocyanin and Eu-8044-streptavidin were then added and followed by one hr incubation at room temperature. Plates were read using the Envision fluorescence reader. IC50 values were determined from inter-plate duplicate or triplicate sets of data. Data were analyzed by XLfit4 (Microsoft) using a 4 Parameter Logistic Model (Sigmoidal Dose-Response Model) and the equation Y = (A + ((B - A)/(1 + ((C/x)^D)))), where A and B were enzyme activity in the absence or presence of infinite inhibitor compound, respectively, C was the IC50 and D was the Hill coefficient.

Cell lines

Wild-type (wt)-p53 cancer cell lines (SJSA1, RKO, HCT116)

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

~ 10 μM; 24 hrs

Applications

In cancer cells expressing wt-p53, RG7388 inhibited cell proliferation with an IC50 value of 30 nM, and induced dose-dependent p53 stabilization, cell cycle arrest, as well as cell apoptosis.

Animal models

Mice bearing SJSA1 human osteosarcoma xenografts

Dosage form

25 or 50 mg/kg; p.o.; q.d., for 32 days

Applications

In a mouse SJSA1 human osteosarcoma xenograft model, RG7388 (25 mg/kg, p.o.) caused tumor growth inhibition and regression.

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.

RG7388 is a second generation clinical MDM2 inhibitor with superior potency and selectivity.
It is a highly potent pyrrolidine compound. RG7388 is more potent and selective than RG7112. In human cancer cell lines, IC50 value of RG7388 in HTRF binding assays is 6 nM and IC50 value of RG7388 in MTT proliferation assays is 0.03μM. [1]
In human cancer cell lines, RG7388 blocks p53−MDM2 binding and effectively activates the p53 pathway, leading to cell cycle arrest and/or apoptosis in cell lines expressing wild-type p53 and tumor growth inhibition or regression of osteosarcoma xenografts in nude mice. RG7388 is undergoing clinical investigation in solid and hematological tumors. [1]
In rhabdomyosarcoma xenografts mice, RG7388 increased the activity of Ionizing radiation (XRT) in both rhabdomyosarcoma models and did not increasing local XRT-induced skin toxicity. Changes in TP53-responsive genes were consistent with the synergistic activity of RG7388 and XRT in the Rh18 model. [2]
RG7388 GI50 concentrations of wt p53 was a >200-fold difference versus mutant cell lines. Comparing with MYCN- cells, Tet21N MYCN+ cells were more sensitive to RG7388. In five p53-wt neuroblastoma cell lines, combining use of RG7388 with cisplatin, topotecan, doxorubicin, busulfan and temozolomide were synergistic led to increased apoptosis and higher caspase-3/7 activity. RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity. [3]
References:
1.Ding Q, Zhang Z, Liu JJ et al. Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development. J Med Chem. 2013 Jul 25;56(14):5979-83.
2.Phelps D, Bondra K, Seum S et al. Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma. Pediatr Blood Cancer. 2015 Apr 1. doi: 10.1002/pbc.25465.
3.Chen L, Rousseau RF, Middleton SA et al. Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma. Oncotarget. 2015 Apr 30;6(12):10207-21.