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

<div><p>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.</p></div

T22 reporter assay measuring p53 transactivation.

<p>A. Cells were treated with increasing concentrations (5, 10, 20 μM) of indicated peptide ligands and p53 activity determined by measuring β galactosidase levels. The small molecule Mdm2 inhibitor Nutlin (10 μM) was used as positive control. Activity is expressed as fold increase over cells treated with DMSO vehicle only. Values represent average ± SD (n = 2). * p < 0.001 compared to DMSO control. B. Lactate dehydrogenase release assay indicating no significant membrane disruption observed for increasing concentrations (2, 10, 20 μM) of indicated ligands compared to vehicle (DMSO) and positive maximum lysis control (MLC). Values represent average ± SD (n = 3).</p

Apparent K_ds (nM) for peptides binding to Mdm2/Mdm4 N-terminal domains and indicated variants.

<p>No significant variations are observed for binding of peptides to Mdm2-M62A (used in structural studies) compared to Mdm2. Values represent average ± SD (n = 2). Previously reported values are 34.35 ± 2.03 (_*), 6.76 ± 2.11(_**), 45.73 ± 7.65 (#) and 1360 ± 600 nM (##) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189379#pone.0189379.ref008" target="_blank">8</a>].</p

Overlay of residues comprising the Trp pocket in M011-Mdm2 and Cpd2-Mdm4 structures.

<p>M011 peptide (green) 6-chloro group of W23 projects deeper into the Mdm4 (cyan) Trp pocket compared to linear Cpd2 peptide (magenta), potentially clashing with the L98 side chain. The position of the corresponding I99 residue in Mdm2 (blue) results in a wider Trp pocket. The mutation of F86 in Mdm2 to L85 in Mdm4 results in the loss of a favorable Van der Waals interaction with the 6-chloro moiety of W23. In addition, the absence of F86 results in a conformational change in the α2 helix between the two proteins. This causes a reorientation of the L98 residue in Mdm4 in comparison to I99 of Mdm2 and brings it into closer proximity to the 6-chloro moiety, forming a less energetically favorable hydrophobic interaction. Several regions of Mdm2/Mdm4 have been omitted for clarity.</p

T22 reporter assay measuring p53 transactivation.

<p>A. Cells were treated with increasing concentrations (5, 10, 20 μM) of indicated peptide ligands and p53 activity determined by measuring β galactosidase levels. The small molecule Mdm2 inhibitor Nutlin (10 μM) was used as positive control. Activity is expressed as fold increase over cells treated with DMSO vehicle only. Values represent average ± SD (n = 2). * p < 0.001 compared to DMSO control. B. Lactate dehydrogenase release assay indicating no significant membrane disruption observed for increasing concentrations (2, 10, 20 μM) of indicated ligands compared to vehicle (DMSO) and positive maximum lysis control (MLC). Values represent average ± SD (n = 3).</p

Structural overlay of M06 and M011 peptides bound to Mdm2.

<p>Only the tryptophan residue of each peptide (with 6-chloro group shown in green for M011) and amino acids forming the Mdm2 Trp pocket are depicted. M06: orange, blue; M011: cyan, magenta. Corresponding Mdm2 residues for each peptide are in the same colour. Image generated using structures 4UMN and 5XXK.</p

Crystallographic data collection and refinement statistics.

<p>Highest resolution bin data stated in parentheses. RMSD values correspond to the root-mean-square deviations of bond lengths and angles of the final restrained and refined structure from experimentally determined ideal values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189379#pone.0189379.ref026" target="_blank">26</a>].</p

Sequence alignment of peptide ligands binding the Mdm2/Mdm4 N-terminal domains.

<p>Shaded residues are the conserved interacting residues (F19, W23 and L26) derived from p53. Xr and Xs represent (<i>S</i>)-2-(4′pentenyl) alanine and (<i>S</i>)-2-(7′-octenyl) alanine (coupled by olefin metathesis).</p

Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design

Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design