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.

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.

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.

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Higher eukaryotic development is a complex and tightly regulated process, whereby transcription factors (TFs) play a key role in controlling the gene regulatory networks. Dysregulation of these regulatory networks has also been associated with carcinogenesis. Transcription factors are key enablers of cancer stemness, which support the maintenance and function of cancer stem cells that are believed to act as seeds for cancer initiation, progression and metastasis, and treatment resistance. One key area of research is to understand how these factors interact and collaborate to define cellular fate during embryogenesis as well as during tumor development. This review focuses on understanding the role of TFs in cell development and cancer. The molecular mechanisms of cell fate decision are of key importance in efforts towards developing better protocols for directed differentiation of cells in research and medicine. We also discuss the dysregulation of TFs and their role in cancer progression and metastasis, exploring TF networks as direct or indirect targets for therapeutic intervention, as well as specific TFs’ potential as biomarkers for predicting and monitoring treatment responses.

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.

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.

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.