Structure of a Stapled Peptide Antagonist Bound to Nutlin-Resistant Mdm2

Abstract

As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

The p53–Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans

“…7A, lanes 4-6). In this regard, Lj-Mdm2 Nterm mimics the recently described M62A mutant of human HDM2 (Chee et al 2014), but the structural basis is in fact distinct. The pocket of HDM2 Nterm /Lj-Mdm2…”

“…Fluorescence anisotropy titrations of purified wild-type HDM2 Nterm (both 1-125 and 6-125) with 50 nM FAM-12.1 (FAM-RFMDYWEGL-NH 2 ) were performed as previously described (Brown et al 2013;Chee et al 2014) to first determine the dissociation constants for the peptide-protein interaction. Subsequent determination of apparent K D s of Nutlin, human, and lamprey peptides was performed by competitive fluorescence anisotropy in which titrations of the peptides were carried out with a constant concentration of HDM2 and FAM-labeled peptide (Brown et al 2013;Chee et al 2014).…”

“…Subsequent determination of apparent K D s of Nutlin, human, and lamprey peptides was performed by competitive fluorescence anisotropy in which titrations of the peptides were carried out with a constant concentration of HDM2 and FAM-labeled peptide (Brown et al 2013;Chee et al 2014). Readings were carried out using the Envision multilabel reader (PerkinElmer).…”

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides

“…Notably, PM2 and several derivatives are able to bind and antagonize Nutlin-resistant HDM2 [45]. This is attributed to the broad, diffuse network of contacts they form with HDM2, which contrasts with the intrinsically limited number of “anchor” points employed by the comparatively small molecule Nutlin [20, 46, 47]. …”

Recognition Dynamics of p53 and MDM2: Implications for Peptide Design

“…One approach to reduce the dimensionality of this search space is to use peptide-based mimics [19][20][21] of protein-protein interfaces that competitively prevent the binding of endogenous biological inhibitors/regulators. High-resolution NMR and crystal structures of MDM2 in complex with a variety of peptides and small molecules are currently available [22][23][24][25][26][27] , providing a plethora of information about their interaction interfaces and providing clues of potential strategies for the development of inhibitors of the p53-MDM2 interaction. Importantly, the crystal structure of the MDM2 protein bound to a 12-residue (E 17 TFSDLWKLLP 27 ) peptide of the p53 trans-activation domain (wild-type p53 peptide) reveals that residues F19, W23 and L26 of the trans-activation domain of p53 are critical for p53 MDM2 association 27 .…”