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Protein–protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers., Stabilising protein–protein interactions is challenging, yet therapeutically important. Native mass spectrometry can be used to monitor binding equilibria, allowing identification and measurement of novel protein–protein interaction stabilisers.

The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure-activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole (FM26) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.

The nuclear receptor RORγt is a key positive regulator in the differentiation and proliferation of T helper 17 (Th17) cells and the production of proinflammatory cytokines like IL-17a. Dysregulation of this pathway can result in the development of various autoimmune diseases, and inhibition of RORγt with small molecules thus holds great potential as a therapeutic strategy. RORγt has a unique allosteric ligand binding site in the ligand binding domain, which is distinct from the canonical, orthosteric binding site. Allosteric modulation of RORγt shows high potential, but the targeted discovery of novel allosteric ligands is highly challenging via currently available methods. Here, we introduce covalent, orthosteric chemical probes for RORγt that occlude the binding of canonical, orthosteric ligands but still allow allosteric ligand binding. Ultimately, these probes could be used to underpin screening approaches for the unambiguous and rapid identification of novel allosteric RORγt ligands.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein-protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like 'molecular glues' is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3o at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3o PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3o, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.

Most cancers evolve to disable the p53 pathway, a key tumour suppressor mechanism that prevents transformation and malignant cell growth. However, only ~50% exhibit inactivating mutations of p53, while in the rest its activity is suppressed by changes in the proteins that modulate the pathway. Therefore, restoring p53 activity in cells in which it is still wild type is a highly attractive therapeutic strategy that could be effective in many different cancer types. To this end, drugs can be used to stabilise p53 levels by modulating its regulatory pathways. However, despite the emergence of promising strategies, drug development has stalled in clinical trials. The need for alternative approaches has shifted the spotlight to the 14-3-3 family of proteins, which strongly influence p53 stability and transcriptional activity through direct and indirect interactions. Here, we present the first detailed review of how 14-3-3 proteins regulate p53, with special emphasis on the mechanisms involved in their binding to different members of the pathway. This information will be important to design new compounds that can reactivate p53 in cancer cells by influencing protein–protein interactions. The intricate relationship between the 14-3-3 isoforms and the p53 pathway suggests that many potential drug targets for p53 reactivation could be identified and exploited to design novel antineoplastic therapies with a wide range of applications.

The demand for allosteric targeting of nuclear receptors is high, but examples are limited, and structural information is scarce. The retinoic acid‐related orphan receptor gamma t (RORγt) is an important transcriptional regulator for the differentiation of T helper 17 cells for which the first, and some of the most promising, examples of allosteric nuclear receptor modulation have been reported and structurally proven. In a 2015 patent, filed by the pharmaceutical company Glenmark, a new class of small molecules was reported that act as potent inverse agonists for RORγt. A compound library around the central thienopyrazole scaffold captured a clear structure‐activity relationship, but the binding mechanism of this new class of RORγt modulators has not been elucidated. Using a combination of biochemical and X‐ray crystallography studies, here the allosteric mechanism for the inverse agonism for the most potent compound, classified in the patent as “example 13”, is reported, providing a strongly desired additional example of allosteric nuclear receptor targeting., Example 13: Inhibition of the RAR‐related orphan receptor gamma t (RORγt) nuclear receptor has proven to be a novel approach for targeting autoimmune diseases. Recently, RORγt was shown to feature an allosteric pocket. Using X‐ray crystallography, we now show that compound 13, a promising thienopyrazole analogue of previously reported RORγt modulators, exerts its inverse agonism by binding to this allosteric pocket.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein–protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like ‘molecular glues’ is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth., The aminothiol WR-1065 covalently modifies 14-3-3σ to stabilse its interactions with p53 and ERα. It enhances the effect of fusicoccin A via a cooperative mechanism that leads to 14-3-3 partner-protein specific activty against cancer cells.

Significance RORγt is a nuclear receptor associated with several diseases. Various synthetic ligands have been developed that target the canonical orthosteric or a second, allosteric pocket of RORγt. We show that orthosteric and allosteric ligands can simultaneously bind to RORγt and that their potency is positively influenced by the other ligand, a phenomenon called cooperative dual ligand binding. The mechanism behind cooperative binding in proteins is poorly understood, primarily due to the lack of structural data. We solved 12 crystal structures of RORγt, simultaneously bound to various orthosteric and allosteric ligands. In combination with molecular dynamics, we reveal a mechanism responsible for the cooperative binding behavior. Our comprehensive structural studies provide unique insights into how cooperative binding occurs in proteins., Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.

International audience; 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 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs.

Binding of 14-3-3 proteins to leucine-rich repeat protein kinase 2 (LRRK2) is known to be impaired by many Parkinson's disease (PD)-relevant mutations. Abrogation of this interaction is connected to enhanced LRRK2 kinase activity, which in turn is implicated in increased ubiquitination of LRRK2, accumulation of LRRK2 into inclusion bodies and reduction in neurite length. Hence, the interaction between 14-3-3 and LRRK2 is of significant interest as a possible drug target for the treatment of PD. However, LRRK2 possesses multiple sites that, upon phosphorylation, can bind to 14-3-3, thus rendering the interaction relatively complex. Using biochemical assays and crystal structures, we characterize the multivalent interaction between these two proteins.

Introduction: PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.

Complementary cyclisation reactions of hex-2-ene-1,6-diamine derivatives were exploited in the synthesis of alternative molecular scaffolds. The value of the synthetic approach was analysed using LLAMA, an open-access computational tool for assessing the lead-likeness and novelty of molecular scaffolds.

An efficient synthetic route to the putative biosynthetic intermediate of the anti-plasmodial natural product janoxepin is described. This novel enamine-containing pyrazino[2,1- b ]quinazoline-3,6-dione, and its synthetic precursors, should be of value in studies to elucidate the biosynthetic pathway leading to the oxepine family of natural products. The cornerstones of the synthesis are amide coupling, pyrazino[2,1- b ]quinazoline-3,6-dione construction and aldol introduction of the enamine.

The total synthesis of (±)-janoxepin, a novel antiplasmodial d-leucine derived oxepine-pyrimidinone-ketopiperazine isolated from the fungus Aspergillus janus, is described. The cornerstones of the synthetic route are pyrimidinone preparation, ring-closing metathesis, aldol introduction of the enamide, and dihydro-oxepine elaboration. This synthetic route proved very efficient for the formation of a number of janoxepin analogues, including dihydro-janoxepin and tetrahydro-janoxepin.

Four amino acid-derived building blocks are converted into 22 scaffolds, e.g. azides (III) and (XIV), hydantoins (IV), lactams (VI), tetrahydropyridines (IX), and azepanes (XII).

A strategy for the efficient lead-oriented synthesis of novel molecular scaffolds is demonstrated. Twenty two scaffolds were prepared from four quaternary α-amino acid building blocks in only 49 synthetic operations, using six connective reactions. The ability of each scaffold to specifically target leadlike chemical space was demonstrated computationally.

Sourcing large numbers of lead-like molecules – compounds that would serve as good starting points for drug discovery programmes – is currently very challenging. The concept of lead-oriented synthesis has recently been articulated to capture the specific problem of preparing diverse small molecules with lead-like molecular properties. In this Feature, some methods that might be used to prepare lead-like molecular scaffolds are described, and presented in the context of diversity-oriented synthetic strategies that allow wide variation in molecular scaffold. It is concluded that the development of a wider toolkit of reactions that is reliable with more polar substrates will be required to allow genuine combination of molecular scaffold within lead-like chemical space.

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.