Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein-Protein Interactions.

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.

Stapled Peptides Inhibitors: A New Window for Target Drug Discovery.

Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach intracellular targets. A novel class -hydrocarbon-stapled α-helical peptides that are synthetic mini-proteins locked into their bioactive structure through site-specific introduction of a chemical linker- has shown promise. Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional small molecule or biologics, suggesting that they offer a novel therapeutic modality. In this review, we highlight what stapling adds to natural-mimicking peptides, describe the revolution of synthetic chemistry techniques and how current drug discovery approaches have been adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM), lactamisation, cycloadditions and reversible reactions. We provide an overview on the available stapled peptide high-resolution structures in the protein data bank, with four selected structures discussed in details due to remarkable interactions of their staple with the target surface. We believe that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to reach currently "undruggable" space.

A fluorinated indole-based MDM2 antagonist selectively inhibits the growth of p53wt osteosarcoma cells.

The p53 protein is engaged in the repair of DNA mutations and elimination of heavily damaged cells, providing anticancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation is often caused by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53 wild-type (p53wt ) cells and thus provides positive outcomes in the treatment of p53wt cancers. Previously, we have reported the discovery of a panel of fluoro-substituted indole-based antagonists of MDM2. Here, we demonstrate the biological activity and stereoselectivity of the most active compound from this series. Both enantiomers of the esterified form of the compound, as well as its corresponding carboxylic acids, were found active in fluorescence polarization (FP) assay, nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) assay, with Ki and KD values around 1 µm. From these four compounds, the esterified enantiomer (R)-5a was active in cells, which was evidenced by the increase of p53 levels, the induced expression of p53-target genes (CDKN1A and MDM2), the selective induction of cell cycle arrest, and selective growth inhibition of p53wt U-2 OS and SJSA-1 compared to p53del SAOS-2 cells. The analysis of the crystal structure of human MDM2 in complex with the compound (R)-6a (carboxylic acid of the active (R)-5a compound) revealed the classical three-finger binding mode. Altogether, our data demonstrate the activity of the compound and provide the structural basis for further structure optimization.

The Pex4p-Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization.

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Šresolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.

Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex.

Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.