Discovery and Characterization of a Novel Series of Chloropyrimidines as Covalent Inhibitors of the Kinase MSK1.

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis.

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.

Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist.

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Structural insights into the disruption of TNF-TNFR1 signalling by small molecules stabilising a distorted TNF.

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization.

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer.

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.

Cryo-EM in drug discovery.

The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.