M. Rossmann

Alternative modulation of protein–protein interactions by small molecules

Graphical abstract

Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.

Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides.

Development of a multipurpose scaffold for the display of peptide loops

Abstract Protein–protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2β/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts.