Abstract 4648: In vitro characterization of spiro-oxindole-based modulators of the MDM2-p53 interaction and their interspecies selectivity.

Abstract

We have identified two spiro-oxindole-based modulators -compounds A and B- of the MDM2-p53 protein-protein interaction that display suitable pharmacological properties for human clinical testing (1) and a high affinity to the N-terminal domain of human MDM2. For instance, in fluorescence polarization (FP) displacement assay using N-terminal domain of human MDM2 and a p53-based peptide (2), compounds A and B display Ki values of 5 and 5,9 nM, respectively, while Nutlin 3 shows a Ki value of 80,3 nM. The superior binding activity of the spiro-oxindole derivatives was confirmed by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Having established their binding activities for the human target and in order to properly address potential on-target adverse effects in preclinical settings, we cloned MDM2 N-terminal domains of monkey, dog, rat and mouse. Sequencing analysis showed that, while the rat data from NCBI Reference Sequence database (3) is inaccurate, there is a high identity between human and monkey sequences. FP displacement assays were developed using MDM2 N-terminal domains of dog, rat and mouse which matched above-mentioned N-terminal domain of human MDM2. Ki value determinations revealed that the binding affinities of compounds A and B but not of Nutlin 3 significantly shifted to higher values by using both rodent MDM2 while, by using canine MDM2, they were similar to the ones determined with human MDM2. These findings were unexpected since MDM2 N-terminal domain sequences are conserved between rodent and non-rodent species. Cellular p53 activation data obtained with Nutlin 3, compounds A and B in a panel of monkey, dog, rat and mouse cell lines were consistent with the biochemical findings and confirmed that compounds A and B are modest p53 activators in rodent cells. In line with the results obtained in cellular settings, acute in vivo induction of p53-dependent transactivation by compounds A and B was significantly weaker in mouse spleen than in human xenografted tumor samples. In conclusion, these data demonstrate that compounds A and B both display high affinity against human MDM2 and that modulators of the MDM2-p53 protein-protein interaction can exhibit inter-species selectivity. These findings should be taken into consideration in the assessment of the tolerabily of MDM2-p53 antagonists in preclinical settings. 1. Wang S. et al., AACR, Orlando FL, 2011, Abstract LB-204 2. Ding K. et al, J. Am. Chem. Soc. 127, 10130-1, 2005. 3. http://www.ncbi.nlm.nih.gov/protein/NP_001101569.1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4648. doi:1538-7445.AM2012-4648

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours

“…data showed that the more potent the MDM2 inhibitor was against human neuroblastoma cells, the less sensitive the murine cells were, and the greater the fold difference between GI 50 values of Trp53 wt murine cells vs. TP53 wt human cells. These observations are consistent with the inter-species selectivity of spiro-oxindole-based MDM2 inhibitors (36) and dihydroisoquinolinone NVP-CGM097 (37), but not pyrazolopyrrolidinone NVP-HDM201 (38,39), and should be taken into account when designing studies of MDM2 inhibitors either alone or in combination using either preclinical transgenic or human tumour xenograft models as p53-dependent normal tissue toxicity will not be adequately modelled. Currently, murine neuroblastoma models include genetically engineered mouse models, syngeneic models, and subcutaneous, orthotopic, pseudometastatic and patient-derived xenografts (3,(40)(41)(42)(43).…”

Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma

“…Future in vivo studies to support observations of the present in vitro study, including dosing and scheduling, will need to be conducted in appropriate in vivo models of both efficacy and toxicity prior to clinical evaluations of RG7388 in neuroblastoma patients. For MDM2-p53 antagonists murine transgenic models may not be suitable as despite a high degree of homology between human and mouse MDM2 [ 33 ], MDM2-p53 antagonists display interspecies selectivity, with reduced binding affinities for mouse and rat MDM2 [ 34 , 35 ]. Consistent with this, we have observed that cell lines derived from MYCN transgenic mice are less sensitive to MDM2-p53 antagonists than human neuroblastoma cell lines ( Chen et al, manuscript in preparation ).…”