Functional display of bioactive peptides on the vGFP scaffold.

Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein-protein interactions.

Structure-activity studies of Mdm2/Mdm4-binding stapled peptides comprising non-natural amino acids.

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6-125) at 1.66 Å resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.

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.