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The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) — EESTSFQGP — as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656–762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM — GKAP — FITC-Ahx-EAQTRL-COOH — the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1. [ABSTRACT FROM AUTHOR]

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis and senescence. The p53 pathway has therefore served as the focus of multiple drug‐discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14‐3‐3 proteins as hDMX and hDM2 client proteins. 14‐3‐3 proteins are adaptor proteins that modulate localization, degradation and interactions of their targets in response to phosphorylation. Thus, 14‐3‐3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript, we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14‐3‐3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20–25 residues apart in both hDMX and hDM2 co‐operate to facilitate high‐affinity 14‐3‐3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts. [ABSTRACT FROM AUTHOR]

Methods to constrain peptides in a bioactive α‐helical conformation for inhibition of protein‐protein interactions represent an ongoing area of investigation in chemical biology. Recently, the first example of a reversible "stapling" methodology was described which exploits native cysteine or homocysteine residues spaced at the i and i + 4 positions in a peptide sequence together with the thiol selective reactivity of dibromomaleimides (a previous study). This manuscript reports on the optimization of the maleimide based constraint, focusing on the kinetics of macrocyclization and the extent to which helicity is promoted with different thiol containing amino acids. The study identified an optimal stapling combination of X1 = L‐Cys and X5 = L‐hCys in the context of the model peptide Ac‐X1AAAX5‐NH2, which should prove useful in implementing the dibromomaleimide stapling strategy in peptidomimetic ligand discovery programmes. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural‐diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity‐directed synthesis (ADS), a function‐driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small‐molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide‐based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia‐inducible factor 1 (HIF‐1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF‐1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α‐helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α‐helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra. [ABSTRACT FROM AUTHOR]

Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue‐unbiased diazirine‐based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol‐containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this "tag and transfer" approach is demonstrated using a well‐defined peptide/protein regulatory interaction (BID80‐102/MCL‐1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA). Fast, enrichable photocrosslinking: Photocrosslinking reagents with methanethiosulfonate and diazirine functionalities have been developed. After crosslinking and reduction, a thiol tag stays at the interaction site, to be located by mass spectrometry and used for enrichment of even low abundance crosslinks. A UV LED irradiation platform reduces crosslinking times to 10 s. [ABSTRACT FROM AUTHOR]

Motivation In experimental protein engineering, alanine-scanning mutagenesis involves the replacement of selected residues with alanine to determine the energetic contribution of each side chain to forming an interaction. For example, it is often used to study protein–protein interactions. However, such experiments can be time-consuming and costly, which has led to the development of programmes for performing computational alanine-scanning mutagenesis (CASM) to guide experiments. While programmes are available for this, there is a need for a real-time web application that is accessible to non-expert users. Results Here, we present BAlaS, an interactive web application for performing CASM via BudeAlaScan and visualizing its results. BAlaS is interactive and intuitive to use. Results are displayed directly in the browser for the structure being interrogated enabling their rapid inspection. BAlaS has broad applications in areas, such as drug discovery and protein-interface design. Availability and implementation BAlaS works on all modern browsers and is available through the following website: https://balas.ap p. The project is open source, distributed using an MIT license and is available on GitHub (https://github.com/wells-wood-research/balas). [ABSTRACT FROM AUTHOR]

A major current challenge in bioorganic chemistry is the identification of effective mimics of protein secondary structures that act as inhibitors of protein-protein interactions (PPIs). In this work, trans-2-aminocyclobutanecarboxylic acid ( tACBC) was used as the key β-amino acid component in the design of α/β/γ -peptides to structurally mimic a native α-helix. Suitably functionalized α/β/γ-peptides assume an α-helix-mimicking 12,13-helix conformation in solution, exhibit enhanced proteolytic stability in comparison to the wild-type α-peptide parent sequence from which they are derived, and act as selective inhibitors of the p53/ hDM2 interaction. [ABSTRACT FROM AUTHOR]

The development of protein–protein interaction (PPIs) inhibitors represents a challenging goal in chemical biology and drug discovery. PPIs are problematic targets because they involve large surfaces with less well defined features and recognition motifs that are less amenable to conventional experimental and computational ligand discovery methodologies. α-Helix mediated PPIs represent a sub group with a clearly defined interface and thus may be more amenable to the development of generic ligand discovery methods. Indeed, this is borne out in numerous studies using peptides covalently constrained into a helical conformation resulting in improvement of myriad biophysical and cellular properties. It is however desirable to have small molecule alternatives: a helix mimetic ( proteomimetic ) is a generic small molecule scaffold that projects functional groups in a similar spatial orientation so as to mimic the presentation of key amino acid side chains from the helix that mediates the PPI. The first true example of a helix mimetic was described over a decade ago however this approach has not yet been elaborated to the extent that it receives similar levels of attention to constrained peptides. This review explores recent significant developments in the area of small molecule α-helix mimetics and provides a critical overview of success stories, potential limitations of the approach, and areas for future development. [ABSTRACT FROM AUTHOR]

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed 'proteomimetics', which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified. [ABSTRACT FROM AUTHOR]

The development of inhibitors of protein-protein interactions (PPIs) represents a major challenge in chemical biology. α-Helix-mediated PPIs represent a subclass that might be amenable to inhibition using scaffolds that reproduce the spatial projection of recognition groups produced by the helix; these ligands are termed proteomimetics. Such a generic scaffold approach requires robust chemistry that can be used to synthesize reasonably large libraries of compounds. Foldamers are defined as oligomers that adopt welldefined folded structures. An ultimate goal of research in this area is to be able to reproduce and surpass the functionality of natural biopolymers; again a key enabling technology in this pursuit is robust synthetic methodology with a broad substrate scope. When we started our research program in this area seven years ago, we were drawn to aromatic oligoamide foldamers as potential proteomimetic scaffolds; however, in contrast to more widely studied β-peptideand peptoid-based foldamers, the synthesis of aromatic oligoamides was less well developed in terms of monomer diversity and amenability to library synthesis. This account describes our efforts and, more generally, the development of methodologies for the synthesis of aromatic oligoamides during this period. [ABSTRACT FROM AUTHOR]

This paper describes the design, synthesis and structural analysis of a 3- O-alkylated aromatic oligoamide that incorporates an additional hydrophilic 6- O-alkyl substituent in the central monomer. This oligomer exhibits low μ M inhibitory potency against the p53- hDM2 interaction compared with its unfunctionalised counterpart and significantly improved solubility. [ABSTRACT FROM AUTHOR]

Rapid access to rigid rods: A method is described for the synthesis of 3‐O‐alkylated aromatic oligobenzamide foldamers that could be used for assembly of libraries of α‐helix mimetic inhibitors of protein–protein interactions (see scheme; Fmoc=9‐fluorenylmethoxycarbonyl). [ABSTRACT FROM AUTHOR]

Inhibition of protein-protein interactions (PPIs) represents a significant challenge because it is unclear how they can be effectively and selectively targeted using small molecules. Achieving this goal is critical given the defining role of these interactions in biological processes. A rational approach to inhibitor design based on the secondary structure at the interface is the focus of much research, and different classes of designed ligands have emerged, some of which effectively and selectively disrupt targeted PPIs. This Review discusses the relevance of PPIs and, in particular, the importance of α-helix-mediated PPIs to chemical biology and drug discovery with a focus on designing inhibitors, including constrained peptides, foldamers and proteomimetic-derived ligands. In doing so, key challenges and major advances in developing generic approaches for the elaboration of PPI inhibitors are highlighted. The challenges faced in developing such ligands as drug leads - and how criteria applied to these may differ from conventional small-molecule drugs - are summarized. [ABSTRACT FROM AUTHOR]

The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix. [ABSTRACT FROM AUTHOR]

Alkenylglycine amino acids were assessed as potential candidates for hydrocarbon stapling and shown to be effective in stapling of the BID BH3 peptide. [ABSTRACT FROM AUTHOR]

[Display omitted] Development of protein–protein interaction (PPI) inhibitors remains a major challenge. A significant number of PPIs are mediated by helical recognition epitopes; although peptides derived from such epitopes are attractive templates for inhibitor design, they may not readily adopt a bioactive conformation, are susceptible to proteolysis and rarely elicit optimal cell uptake properties. Constraining peptides has therefore emerged as a useful method to mitigate against these liabilities in the development of PPI inhibitors. Building on our recently reported method for constraining peptides by reaction of dibromomaleimide derivatives with two cysteines positioned in an i and i + 4 relationship, in this study, we showcase the power of the method for rapid identification of ideal constraining positions using a maleimide-staple scan based on a 19-mer sequence derived from the BAD BH3 domain. We found that the maleimide constraint had little or a detrimental impact on helicity and potency in most sequences, but successfully identified i , i + 4 positions where the maleimide constraint was tolerated. Analyses using modelling and molecular dynamics (MD) simulations revealed that the inactive constrained peptides likely lose interactions with the protein as a result of introducing the constraint. [ABSTRACT FROM AUTHOR]

Abstract: Protein–protein interactions (PPIs) mediate cellular pathways and are implicated in numerous aberrant conditions. α-Helix mimetics—small molecules that reproduce the spatial projection of key residues from an α-helix involved in a PPI—are attractive generic templates for development of screening libraries, however library syntheses of α-helix mimetics with diverse functionality are less established. This manuscript describes the automated, microwave assisted solid phase synthesis based on one such scaffold; an N-alkylated oligobenzamide. [Copyright &y& Elsevier]

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as "undruggable" targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products. [ABSTRACT FROM AUTHOR]

Abstract: In this article, we report the synthesis, solid-state and solution-state conformational studies of O-alkylated aromatic benzamides based on two scaffolds. Intramolecular hydrogen bonding provides conformational pre-organization and side chains can interact with each other within a molecule. In the solid-state three-dimensional arrangement, the molecules further interact with each other through non-covalent interactions. Given, the demonstrated potential of this class of scaffolds to act as helix mimetics for the inhibition of protein–protein interactions (PPIs), these results provide key insight for future inhibitor design. [Copyright &y& Elsevier]

Abstract: This manuscript describes cell-uptake studies with HEK 293T cells on a series of ruthenium complexes shown previously to act as receptors for protein surface recognition and was motivated by a desire to establish if these receptors represent suitable templates for further elaboration as inhibitors of protein–protein interactions. The results illustrate that large (>3000Da) highly functionalized anionic ruthenium complexes are efficiently transfected via endocytosis to lysosomes with negligible toxicity. [Copyright &y& Elsevier]

Protein-protein interactions (PPIs) play a critical role in living cells and represent promising targets for the drug discovery and life sciences communities. However, lateral transmembrane PPIs are difficult targets for small-molecule inhibitor development given less structural information is known and fewer ligand discovery methods have been explored compared to soluble proteins. In this study, the interactions of the transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) were disrupted by pentamidine derivatives to curb the committed step of EBV infection. A pentamidine derivative 2 with a 7-atom di-amide linker had the best activity whilst switching the amide regiochemistry in the linker influenced membrane permeability and abolished anti TMD-5 activity. Molecular dynamics simulations were performed to understand the interaction between pentamidine derivatives and TMD-5, and to rationalise the observed structure-activity relationships. This study explicitly demonstrated that the interaction of small molecule with lipid should be considered alongside interaction with the protein target when designing small molecules targeting the PPIs of TMDs. In all, this study provides proof of concept for the rational design of small molecules targeting transmembrane PPIs. Image 1 • Protein-protein interactions are vital in cells and represent promising target. • Protein-protein interfaces are difficult to target due to inherent challenges. • Compound 2 successfully targeted the interfaces and addressed this challenge. • The activity of compound 2 is extremely influenced by the regiochemistry of linker. • Interactions of molecule with lipid are vital when targeting the interfaces. [ABSTRACT FROM AUTHOR]