Dissecting the functional behavior of the differentially phosphorylated prolyl isomerase, Pin1.

Protein post-translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build-up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis-regulation is prevalent. Here, we focus on Pin1, a peptidyl-prolyl cis-trans isomerase whose regulatory function is altered by a diverse range of PTMs.

Harnessing the 14-3-3 protein-protein interaction network.

Protein-protein interactions (PPIs) play a critical role in cellular signaling and represent interesting targets for therapeutic intervention. 14-3-3 proteins integrate many signaling targets via PPIs and are frequently implicated in disease, making them intriguing drug targets. Here, we review the recent advances in the 14-3-3 field.

Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity.

Tracking the mechanism of covalent molecular glue stabilization using native mass spectrometry.

Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms.

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions.

14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development.

Cooperative stabilisation of 14-3-3σ protein-protein interactions covalent protein modification.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes 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.

Discovering protein-protein interaction stabilisers by native mass spectrometry.

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.

Structure-Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt.

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.

Covalent Occlusion of the RORγt Ligand Binding Pocket Allows Unambiguous Targeting of an Allosteric Site.

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.

Cooperativity between the orthosteric and allosteric ligand binding sites of RORγt.

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.

Regulation of p53 by the 14-3-3 protein interaction network: new opportunities for drug discovery in cancer.

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.

Fragment-based Differential Targeting of PPI Stabilizer Interfaces.

Stabilization of protein-protein interactions (PPIs) holds great potential for therapeutic agents, as illustrated by the successful drugs rapamycin and lenalidomide. However, how such interface-binding molecules can be created in a rational, bottom-up manner is a largely unanswered question. We report here how a fragment-based approach can be used to identify chemical starting points for the development of small-molecule stabilizers that differentiate between two different PPI interfaces of the adapter protein 14-3-3.

Elucidation of an Allosteric Mode of Action for a Thienopyrazole RORγt Inverse Agonist.

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.

Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists.

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.

Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ.

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.

Synthesis and Demonstration of the Biological Relevance of sp -rich Scaffolds Distantly Related to Natural Product Frameworks.

The productive exploration of chemical space is an enduring challenge in chemical biology and medicinal chemistry. Natural products are biologically relevant, and their frameworks have facilitated chemical tool and drug discovery. A "top-down" synthetic approach is described that enabled a range of complex bridged intermediates to be converted with high step efficiency into 26 diverse sp -rich scaffolds.

Modulators of 14-3-3 Protein-Protein Interactions.

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.

Stabilization of protein-protein interactions in drug discovery.

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.

Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

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.

Structural interface between LRRK2 and 14-3-3 protein.

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.

A divergent synthetic approach to diverse molecular scaffolds: assessment of lead-likeness using LLAMA, an open-access computational tool.

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.

A systematic approach to diverse, lead-like scaffolds from α,α-disubstituted amino acids.

A powerful strategy for the efficient lead-oriented synthesis of novel molecular scaffolds is demonstrated. Twenty two scaffolds were prepared from just four α-amino acid-derived building blocks and a toolkit of six connective reactions. Importantly, each individual scaffold has the ability to specifically target lead-like chemical space.

A unified lead-oriented synthesis of over fifty molecular scaffolds.

Controlling the properties of lead molecules is critical in drug discovery, but sourcing large numbers of lead-like compounds for screening collections is a major challenge. A unified synthetic approach is described that enabled the synthesis of 52 diverse lead-like molecular scaffolds from a minimal set of 13 precursors. The divergent approach exploited a suite of robust, functional group-tolerant transformations.

Total synthesis of an oxepine natural product, (±)-janoxepin.

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.