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.

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.

Development of small molecules targeting the PD-L1/PD-1 interface is advancing both in industry and academia, but only a few have reached early-stage clinical trials. Here, we take a closer look at the general druggability of PD-L1 using in silico hot spot mapping and nuclear magnetic resonance (NMR)-based characterization. We found that the conformational elasticity of the PD-L1 surface strongly influences the formation of hot spots. We deconstructed several generations of known inhibitors into fragments and examined their binding properties using differential scanning fluorimetry (DSF) and protein-based nuclear magnetic resonance (NMR). These biophysical analyses showed that not all fragments bind to the PD-L1 ectodomain despite having the biphenyl scaffold. Although most of the binding fragments induced PD-L1 oligomerization, two compounds, TAH35 and TAH36, retain the monomeric state of proteins upon binding. Additionally, the presence of the entire ectodomain did not affect the binding of the hit compounds and dimerization of PD-L1. The data demonstrated here provide important information on the PD-L1 druggability and the structure–activity relationship of the biphenyl core moiety and therefore may aid in the design of novel inhibitors and focused fragment libraries for PD-L1.

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal "lid" segment of MDM2 from the proximity of the p53complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.