The modulation of protein–protein interactions (PPIs) has been recognized as one of the most challenging tasks in drug discovery. While their systematic development has long been considered as ...intractable, this view has changed over the last years, with the first drug candidates undergoing clinical studies. To date, the vast majority of PPI modulators are interaction inhibitors. However, in many biological contexts a prolonged lifespan of a PPI might be desirable, calling for the complementary approach of PPI stabilization. In fact, nature offers impressive examples of this concept and some PPI‐stabilizing natural products have already found application as important drugs. Moreover, directed small‐molecule stabilization has recently been demonstrated. Therefore, it is time to take a closer look at the constructive side of modulating PPIs.
Doing it the other way round: The modulation of protein–protein interactions (PPIs) by small molecules has become increasingly popular over the last few decades. However, “modulation” has mainly been perceived as “inhibition” of protein–protein interactions, omitting the complementary strategy of stabilizing such macromolecular complexes. This Minireview highlights amazing examples and the potential of this constructive side of modulating PPIs.
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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.
14-3-3: A Case Study in PPI Modulation Ballone, Alice; Centorrino, Federica; Ottmann, Christian
Molecules (Basel, Switzerland),
06/2018, Volume:
23, Issue:
6
Journal Article
Peer reviewed
Open access
In recent years, targeting the complex network of protein⁻protein interactions (PPIs) has been identified as a promising drug-discovery approach to develop new therapeutic strategies. 14-3-3 is a ...family of eukaryotic conserved regulatory proteins which are of high interest as potential targets for pharmacological intervention in human diseases, such as cancer and neurodegenerative and metabolic disorders. This viewpoint is built on the “hub” nature of the 14-3-3 proteins, binding to several hundred identified partners, consequently implicating them in a multitude of different cellular mechanisms. In this review, we provide an overview of the structural and biological features of 14-3-3 and the modulation of 14-3-3 PPIs for discovering small molecular inhibitors and stabilizers of 14-3-3 PPIs.
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High-diversity genetically-encoded combinatorial libraries (10
-10
members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access ...broader chemical space, but typically examine only ~ 10
compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10
members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10
-10
. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.
•Protein–protein interactions (PPIs) are ever increasingly targets for drug discovery.•Targeted small molecule inhibition of PPIs has repeatedly been shown to be successful.•Targeted small molecule ...stabilization of PPIs is however an underexplored concept.•We review the current status of small molecule PPI stabilization as a fruitful concept.
Protein–protein interactions (PPIs) are implicated in every disease and mastering the ability to influence PPIs with small molecules would considerably enlarge the druggable genome. Whereas inhibition of PPIs has repeatedly been shown to work successfully, targeted stabilization of PPIs is underrepresented in the literature. This is all the more surprising because natural products like FK506, rapamycin, brefeldin, forskolin and fusicoccin confer their physiological activity by stabilizing specific PPIs. However, recently a number of very interesting synthetic molecules have been reported from drug discovery projects that indeed achieve their desired activities by stabilizing either homo- or hetero-oligomeric complexes of their target proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
Abstract
The mineralized collagen fibril is the basic building block of bone, and is commonly pictured as a parallel array of ultrathin carbonated hydroxyapatite (HAp) platelets distributed ...throughout the collagen. This orientation is often attributed to an epitaxial relationship between the HAp and collagen molecules inside 2D voids within the fibril. Although recent studies have questioned this model, the structural relationship between the collagen matrix and HAp, and the mechanisms by which collagen directs mineralization remain unclear. Here, we use XRD to reveal that the voids in the collagen are in fact cylindrical pores with diameters of ~2 nm, while electron microscopy shows that the HAp crystals in bone are only uniaxially oriented with respect to the collagen. From in vitro mineralization studies with HAp, CaCO
3
and γ-FeOOH we conclude that confinement within these pores, together with the anisotropic growth of HAp, dictates the orientation of HAp crystals within the collagen fibril.
Interactions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14‐3‐3 adapter proteins are a prominent example and typically bind partner proteins ...in a phosphorylation‐dependent mono‐ or bivalent manner. Herein we describe the development of a cucurbit8uril (Q8)‐based supramolecular system, which in conjunction with the 14‐3‐3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine–glycine–glycine (FGG) tripeptide motif to the N‐terminus of the 14‐3‐3‐binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8‐induced dimerization of the ERα epitope augmented its affinity towards 14‐3‐3 through a binary bivalent binding mode. The crystal structure of the Q8‐induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8.
Binary bivalent binding: The combination of a bivalent cucurbit8uril (Q8) host–guest complex with the bivalent protein 14‐3‐3 generated a binary assembly platform. Supramolecular induced switching between mono‐ and bivalent protein modes was observed and the elucidation of the first Q8‐protein cocrystal structure reported.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
The systematic stabilization of protein–protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current ...lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.
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.
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