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An allosteric inhibitor, EAI045, is reported that is selective for certain drug-resistant EGFR mutants, but spares the wild-type receptor; combination therapy of EAI045 with EGFR-dimerization-blocking antibodies is effective in mouse models of lung cancer driven by mutant versions of EGFR that are resistant to all previously developed inhibitors. Novel EGFR-directed therapeutics Currently available small-molecule inhibitors targeting epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases bind the ATP site of the kinase, and therefore typically inhibit a number of 'off-target' kinases owing to the high conservation of this site. In addition, the common binding site of these drugs leads to shared susceptibility to resistance-conferring mutations in EGFR. Here, Michael Eck and colleagues describe an allosteric inhibitor, EAI045, that is selective for certain drug-resistant EGFR mutants but spares the wild-type receptor. Although EAI045 is not effective in blocking EGFR-driven cell proliferation as a single agent, it has synergistic inhibitory activity when combined with an antibody that blocks EGFR dimerization. This combination therapy is effective in mouse models of lung cancer driven by mutant versions of EGFR that are resistant to all previously developed inhibitors. The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase 1 , 2 , but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor 3 , 4 . Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant 5 , 6 , but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond 7 . All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state 8 . We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization 9 , 10 , rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants ( S )-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of ( S )-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design. X-ray crystal structures of the human serotonin transporter (SERT) bound to the antidepressants ( S )-citalopram or paroxetine show that the antidepressants lock the protein in an outward-open conformation, and directly block serotonin from entering its binding site; the structures define the mechanism of antidepressant action in SERT and pave the way for future drug design. Antidepressant structure/activity relationships Serotonin modulates the activity of the central nervous system, as well as many other processes throughout the body. These authors have solved X-ray structures of the human serotonin transporter (SERT) in complex with the selective serotonin reuptake inhibitors (SSRIs) ( S )-citalopram and paroxetine — two of the most widely prescribed antidepressants. The resulting structures reveal that the antidepressants lock the protein in an outward-open conformation, and directly block the entry of serotonin into its binding site. A previously unknown allosteric site is seen in the extracellular vestibule; binding of ligands to this site prevents dissociation from the central site, establishing a mechanism of antidepressant action in SERT and pointing the way for future drug design.

A new mass-spectrometry method has been developed to obtain high-resolution spectra of folded proteins bound to lipids; using this technique as well as X-ray crystallography provides evidence for membrane protein conformational change as a result of lipid–protein interaction. Lipid bound to influence protein structure Many of the high-resolution membrane protein structures published recently are notable for the presence of lipids closely associated with the protein, prompting the question, how are these lipids influencing membrane complex structure? Carol Robinson and colleagues have developed a new ion mobility mass spectrometry (IM-MS) method that enabled them to obtain mass spectra of folded protein conformations bound to lipids. Using this method they identified lipids that altered the stability of MscL (mechanosensitive channel of large conductance), aquaporin Z and the ammonia channel. They then determined the X-ray crystal structure of the ammonia channel bound to one of these lipids (phosphatidylglycerol), which revealed how a conformational change in a specific loop led to the formation of a phosphatidylglycerol-binding site. The major conclusion from this work is that an individual lipid-binding event can change the stability of a membrane complex. On the cover, IM-MS captures a native membrane protein complex emerging from an ion mobility cell. Shown is the ammonia channel in apo, one- and two-lipid bound states. Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments 1 , 2 , 3 , 4 , 5 , 6 , 7 and that lipids can bind to specific sites, for example, in potassium channels 8 . Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli , using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation 9 . AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics. High-resolution structural studies of GPCRs have led to insights into the role of allostery in GPCR-mediated signal transduction.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development 1 – 5 . This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development. A new specific, small-molecule activator of the PI3Kα isoform (UCL-TRO-1938) identified through high-throughput screening can transiently activate PI3K signalling and biological responses in cells and tissues, with potential therapeutic applications in tissue protection and regeneration.

Lung cancer has a high prevalence, with a growing number of new cases and mortality every year. Furthermore, the survival rate of patients with non-small-cell lung carcinoma (NSCLC) is still quite low in the majority of cases. Despite the use of conventional therapy such as tyrosine kinase inhibitor for Epidermal Growth Factor Receptor (EGFR), which is highly expressed in most NSCLC cases, there was still no substantial improvement in patient survival. This is due to the drug’s ineffectiveness and high rate of resistance among individuals with mutant EGFR. Therefore, the development of new inhibitors is urgently needed. Understanding the EGFR structure, including its kinase domain and other parts of the protein, and its activation mechanism can accelerate the discovery of novel compounds targeting this protein. This study described the structure of the extracellular, transmembrane, and intracellular domains of EGFR. This was carried out along with identifying the binding pose of commercially available inhibitors in the ATP-binding and allosteric sites, thereby clarifying the research gaps that can be filled. The binding mechanism of inhibitors that have been used clinically was also explained, thereby aiding the structure-based development of new drugs.

Stabilization of an active and inactive conformation of the β 2 -adrenergic receptor by allosteric nanobodies reveals differential ligand-dependent regulation of receptor states to control G-protein-coupled receptor activation. Agonist binding to the β2-adrenergic receptor In this manuscript, the authors studied how a positive allosteric nanobody (Nb80) and a newly discovered negative allosteric nanobody (Nb60) alter the structure of the β2-adrenergic receptor (β 2 AR). Their data support a three-state model for receptor activation in this important G-protein-coupled receptor, rather than a simple inactive–active two-state model. They also find that full agonists primarily stabilize the active Nb80-stabilized receptor state (while having negligible effects on the inactive Nb60-bound state), but partial agonists appear to regulate multiple receptor states to control receptor activation. G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’) 1 . Furthermore, increasing biophysical evidence, primarily using the β 2 -adrenergic receptor (β 2 AR) as a model system, supports the existence of multiple active and inactive conformational states 2 , 3 , 4 , 5 . However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β 2 AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) 6 , 7 and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β 2 AR in the presence of Nb80 compared to the affinity of isoprenaline for β 2 AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β 2 AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R 80 ). In contrast, for a number of partial agonists, both stabilization of R 80 and destabilization of the inactive, Nb60-bound state (R 60 ) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Here, pharmacological and biochemical evidence is provided that shows that G-protein coupling to the β 2 -adrenergic receptor stabilizes a ‘closed’ conformation of the G-protein-coupled receptor (GPCR) and that that the effects of the G protein on the ligand-binding site of the GPCR are observed even in the absence of a bound agonist. Agonist binding in GPCRs Signalling via G-protein-coupled receptors (GPCRs) is the primary mechanism by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these membrane proteins interact with intracellular G proteins to regulate downstream second messenger and/or protein kinase cascades. This study presents pharmacological and biochemical evidence to shows that G-protein coupling to the β 2 -adrenergic receptor (β 2 AR) stabilizes a 'closed' conformation of the GPCR. The effects of the G protein on the ligand-binding site of the GPCR are observed even in the absence of a bound agonist. This means that binding of the G protein to the GPCR can prevent any ligand — agonists, partial agonists, antagonists and inverse agonists — from interacting with the GPCR. Ligands that have already bound to the GPCR are unable to dissociate from the receptor when the G protein is present. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other 1 . Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades 1 . Crystallographic evidence from a prototypic GPCR, the β 2 -adrenergic receptor (β 2 AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins 2 . The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β 2 AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p -cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.

G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the β adrenergic receptor (β AR) that was recently isolated from a DNA-encoded small-molecule library. Orthosteric β-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the β AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the β AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.