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

Abstract

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.

A benzene-mapping approach for uncovering cryptic pockets in membrane-bound proteins

“…are shown as orange van der Waals spheres with dummy atoms indicated in tan. 18 The success of probe-mapping methods is customarily measured by its ability to increase solvent-accessible surface area (SASA) of the protein, which is partly a result of the favourable exposure of cryptic pockets to the interacting solvent 61 . The hydrophobic character of a probe increases the probability that, upon binding, the pocket -often hidden inside the protein interior -opens up.…”

“…However, conventional MD simulations, even when paired with long simulation timescales or improved sampling methods, are not yet routinely able to reliably detect the full repertoire of transient binding pockets on protein targets 16 . Cryptic pockets are 5 usually hydrophobic in nature, making their opening and exposure to the highly polar water environment energetically unfavourable, and thus under-sampled by the simulated system 17,18 .…”

Macrocyclization of an all-D linear peptide improves target affinity and imparts cellular activity: A novel stapled α-helical peptide modality

“…The three critical residues 6-F-d Trp3, p-CF3-d Phe7 and d Leu11 remained buried in the hydrophobic pocket/binding site of Mdm2 [ Figure 6]. The hydrocarbon linkers remained largely exposed to solvent without engaging the Mdm2 surface; this contrasts with some of the L-amino acid stapled peptides where the staples contributed to the binding by engaging with the surface of Mdm2 [33,52].…”

“…Unfortunately, this peptide lacked cell permeability, but did activate p53 in cells when delivered using nano-carriers [42]. Given our recent success in the cellular inhibition of Mdm2 and activation of p53 by stapled peptides (containing L-amino acids) [33,[51][52][53], we questioned whether we could achieve cellular permeation by stapling the D-peptide. We report here the computational design, synthesis and biological evaluation of stapled versions of d PMIδ.…”

Inhibiting mechanism of small molecule toward the p53‐MDM2 interaction: A molecular dynamic exploration

“…was optimized to engender small molecular compounds with the inhibitory intensity of nM level on the p53‐MDM2 association . Currently, to more efficiently active function of the wild‐type p53, many efforts have been paid to design and develop potent nonpeptide and low molecular weight inhibitors that competitively occupy the binding cleft of MDM2 to efficiently block the p53‐MDM2 interaction …”