David Bier

Constrained peptides with target-adapted cross-links as inhibitors of a pathogenic protein-protein interaction.

Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein-protein interactions (PPI). However, most peptide-derived PPI inhibitors involve stabilized α-helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross-links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X-ray crystallography and isothermal titration calorimetry. The resulting cross-linked peptides inhibit the interaction between human adaptor protein 14-3-3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide-derived inhibitors.

Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T594) for high-affinity interaction. We confirm that T594 is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594–specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.

Small-molecule stabilization of the 14-3-3/gab2 protein-protein interaction (PPI) interface

Small‐molecule modulation of protein–protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under‐represented. Here, we report the example of a semi‐synthetic natural product derivative—ISIR‐005—that stabilizes the cancer‐relevant interaction of the adaptor protein 14‐3‐3 and Gab2. The crystal structure of ISIR‐005 in complex with 14‐3‐3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim‐of‐the‐interface of the protein complex. Only in the direct vicinity of 14‐3‐3/Gab2pT391 site is a pre‐formed pocket occupied by ISIR‐005; binding of the Gab2pS210 motif to 14‐3‐3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR‐005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14‐3‐3/Gab2 PPI interface with important implications for the development of PPI stabilizers.

Identification of a Selective, Activity-Based Probe for Glyceraldehyde 3-Phosphate Dehydrogenases†

Exceptionally specific: The natural-product-like structural complexity of a bicyclic hydantoin was exploited to generate the novel, highly specific activity-based profiling probe (ABPP) Mrl-Rh (Rh=rhodamine; see picture) for glyceraldehyde 3-phosphate dehydrogenases. This probe can be used to investigate activity changes of this enzyme class during plant–pathogen interactions.

Constraining an Irregular Peptide Secondary Structure through Ring-Closing Alkyne Metathesis

Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α‐helices, which limits their general applicability. Here we report for the first time on the use of ring‐closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne‐crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest‐affinity derivative in a complex with its protein target 14‐3‐3ζ. It can be expected that the alkyne‐based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins.

Redox Modulation of PTEN Phosphatase Activity by Hydrogen Peroxide and Bisperoxidovanadium Complexes

PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2O2) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions.

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein–Protein Interface

Protein regions that are involved in protein–protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein–protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular “Janus” ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein–protein interface, “freezes” one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

Structure-Based Design of Non-Natural Macrocyclic Peptides that Inhibit Protein-Protein Interactions

Macrocyclic peptides can interfere with challenging biomolecular targets including protein–protein interactions. Whereas there are various approaches that facilitate the identification of peptide-derived ligands, their evolution into higher affinity binders remains a major hurdle. We report a virtual screen based on molecular docking that allows the affinity maturation of macrocyclic peptides taking non-natural amino acids into consideration. These macrocycles bear large and flexible substituents that usually complicate the use of docking approaches. A virtual library containing more than 1400 structures was screened against the target focusing on docking poses with the core structure resembling a known bioactive conformation. Based on this screen, a macrocyclic peptide 22 involving two non-natural amino acids was evolved showing increased target affinity and biological activity. Predicted binding modes were verified by X-ray crystallography. The presented workflow allows the screening of large macrocyclic peptides with diverse modifications thereby expanding the accessible chemical space and reducing synthetic efforts.

Rational Design, Binding Studies, and Crystal‐Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14‐3‐3ζ Adapter Protein

14‐3‐3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14‐3‐3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14‐3‐3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X‐ray crystallography.