Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 Å resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.

We report a standardized complex molecule diagnostic approach using collections of relevant drug-like molecules which we call chemistry informer libraries.

Since the advent of high-throughput screening (HTS), there has been an urgent need for methods that facilitate the interrogation of large-scale chemical biology data to build a mode of action (MoA) hypothesis. This can be done either prior to the HTS by subset design of compounds with known MoA or post HTS by data annotation and mining. To enable this process, we developed a tool that compares compounds solely on the basis of their bioactivity: the chemical biological descriptor "high-throughput screening fingerprint" (HTS-FP). In the current embodiment, data are aggregated from 195 biochemical and cell-based assays developed at Novartis and can be used to identify bioactivity relationships among the in-house collection comprising ~1.5 million compounds. We demonstrate the value of the HTS-FP for virtual screening and in particular scaffold hopping. HTS-FP outperforms state of the art methods in several aspects, retrieving bioactive compounds with remarkable chemical dissimilarity to a probe structure. We also apply HTS-FP for the design of screening subsets in HTS. Using retrospective data, we show that a biodiverse selection of plates performs significantly better than a chemically diverse selection of plates, both in terms of number of hits and diversity of chemotypes retrieved. This is also true in the case of hit expansion predictions using HTS-FP similarity. Sets of compounds clustered with HTS-FP are biologically meaningful, in the sense that these clusters enrich for genes and gene ontology (GO) terms, showing that compounds that are bioactively similar also tend to target proteins that operate together in the cell. HTS-FP are valuable not only because of their predictive power but mainly because they relate compounds solely on the basis of bioactivity, harnessing the accumulated knowledge of a high-throughput screening facility toward the understanding of how compounds interact with the proteome.

Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

Recent work has shown that the incorporation of an all-hydrocarbon "staple" into peptides can greatly increase their α-helix propensity, leading to an improvement in pharmaceutical properties such as proteolytic stability, receptor affinity and cell-permeability. Stapled peptides thus show promise as a new class of drugs capable of accessing intractable targets such as those that engage in intracellular protein-protein interactions. The extent of α-helix stabilization provided by stapling has proven to be substantially context dependent, requiring cumbersome screening to identify the optimal site for staple incorporation. In certain cases, a staple encompassing one turn of the helix (attached at residues i and i+4) furnishes greater helix stabilization than one encompassing two turns (i,i+7 staple), which runs counter to expectation based on polymer theory. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples. Here we report all-atom Monte Carlo folding simulations comparing unmodified peptides derived from RNAse A and BID BH3 with various i,i+4 and i,i+7 stapled versions thereof. The results of these simulations were found to be in quantitative agreement with experimentally determined helix propensities. We also discovered that staples can stabilize quasi-stable decoy conformations, and that the removal of these states plays a major role in determining the helix stability of stapled peptides. Finally, we critically investigate why our method works, exposing the underlying physical forces that stabilize stapled peptides.