Inhibition of Oncogenic MdmX by Targeting its Motion

Ban, David

Abstract

The function of p53 is abrogated due to missense mutation in ~50% of all human cancers. In others, p53 regulatory pathways are altered. For the pediatric cancer, retinoblastomas, St. Jude (SJ) researchers showed that tumor progression is driven by over-expression of the MdmX protein, a negative p53 regulator. Therefore, inhibition of MdmX can restore p53 function and trigger apoptosis and/or cell arrest. A drug discovery program at SJ is developing inhibitors that target a pocket on the N-terminal domain of MdmX (MdmX-N) which binds to the transactivation domain of p53 (p53 TAD1) with an affinity of ~100 nM. Small molecules called Nutlins bind to the homologous protein Mdm2 and strongly inhibit its interactions with p53 TAD1 (Nutlin 3a, IC50 = ~20 nM). We reasoned that this scaffold could be applied to MdmX-N. However, Nutlin 3a, is a poor inhibitor (IC50 = ~20 mM). To overcome this, the SJ team synthesized a series of Nutlin analogs but achieved only modest gains in potency (IC50 = ~1 mM). The solution structures of these compounds bound to MdmX were similar to the crystal structure of Nutlin 3a bound to Mdm2, providing little insight into structure-activity relationships. Why do the current SJ Nutlin analogs exhibit relatively low affinity for MdmX-N and what strategy can be used to attain an affinity <100 nM? Importantly, our preliminary data show that MdmX-N is highly dynamic. Therefore, we hypothesize that considering only static structural features is insufficient for high affinity compound design. Instead, dynamic features must be quantified and this knowledge used for future compound optimization.
Our Specific Aims are as follows: (1) to define the dynamic landscape of MdmX-N in complex with SJ compounds and a dynamic reference, p53 TAD1 and (2) to understand the dynamics in apo MdmX-N and their influence on interactions with the p53 TAD1 peptide and SJ compounds. Affinities and thermodynamics of the interactions will be determined with Isothermal Titration Calorimetry and NMR based titrations. We will use NMR to detect dynamics in MdmX-N because observables are sensitive to motion between picoseconds to real time and are attained with atomic resolution. MdmX-N's dynamics will be mapped with a battery of fast (ps-ns) and slow (?s-ms) motion experiments that will be used to measure the kinetic and structural details of dynamic state(s) for complexes with all SJ compounds and p53 TAD1. A quantitative comparison of all complexes will be used to identify how compounds can be optimized to mimic dynamic properties of the MdmX-N/p53 TAD1 interaction. These results will guide the synthesis of novel Nutlin analogs in collaboration with Dr. Kip Guy at St. Jude. Furthermore, for NMR studies of apo MdmX-N, systematic screens of conditions (pH, ionic strength, osmolytes) by vapor diffusion, Thermal Shift, and NMR will be performed. Using methods described above a correlation between apo and bound state motions will be built to understand how they affect compound affinities. This work will not only establish a new paradigm by using structure/dynamic-function relationships to guide drug discovery, but will also advance our translational goals to combat retinoblastoma.

Public Health Relevance

MdmX is overexpressed in ~60% of retinoblastomas (affecting exclusively children) and has been demonstrated at St. Jude to contribute to tumorigenesis and tumor progression. Furthermore, MdmX overexpression inactivates p53 in retinoblastoma cells and proof-of-principle studies have shown that inhibition of MdmX/p53 interactions restores p53 activity and caused retinoblastoma cells to die. However, the best MdmX inhibitors available to date (synthesized at St. Jude) exhibit moderate inhibitory abilities; through this proposal, we seek to develop more potent inhibitors by targeting the motion of MdmX for the treatment of retinoblastoma patients.