Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction.

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2A Receptor.

In this study, we determined the crystal structure of an engineered human adenosine A2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.

Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists.

We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.

Towards high throughput GPCR crystallography: In Meso soaking of Adenosine A2A Receptor crystals.

Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity "carrier" ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).

X-ray free electron laser: opportunities for drug discovery.

Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization. To further capitalize on the XFEL advantage, innovations in crystal sample delivery for the X-ray experiment, data collection and processing methods are required. This development was a key contributor to serial crystallography allowing structure determination at room temperature yielding physiologically more relevant structures. Adding the time resolution provided by the femtosecond X-ray pulse will enable monitoring and capturing of dynamic processes of ligand binding and associated conformational changes with great impact to the design of candidate drug compounds.

Structures of Human A1 and A2A Adenosine Receptors with Xanthines Reveal Determinants of Selectivity.

The adenosine A1 and A2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A1 receptor at 3.3 Å resolution with PSB36, an A1-selective xanthine-based antagonist. This is compared with structures of the A2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A1R and A2AR were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T2707.35 in A1R (M2707.35 in A2AR) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A1R or A2AR antagonists.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Structure-based discovery of LpxC inhibitors.

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein.

Decoding Corticotropin-Releasing Factor Receptor Type 1 Crystal Structures.

The structural analysis of class B G protein-coupled receptors (GPCR), cell surface proteins responding to peptide hormones, has until recently been restricted to the extracellular domain (ECD). Corticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor mediating stress response and also considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of human CRF1R in complex with the small-molecule antagonist CP-376395 in a hexagonal setting with translational non-crystallographic symmetry. Molecular dynamics and metadynamics simulations on this novel structure and the existing TMD structure for CRF1R provides insight as to how the small molecule ligand gains access to the induced-fit allosteric binding site with implications for the observed selectivity against CRF2R. Furthermore, molecular dynamics simulations performed using a full-length receptor model point to key interactions between the ECD and extracellular loop 3 of the TMD providing insight into the full inactive state of multidomain class B GPCRs.

Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

Fragment-based discovery of DNA gyrase inhibitors targeting the ATPase subunit of GyrB.

Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.

Structure of class B GPCR corticotropin-releasing factor receptor 1.

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

Discovery and structure-activity relationship of potent and selective covalent inhibitors of transglutaminase 2 for Huntington's disease.

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

HCV NS5B polymerase inhibitors 2: Synthesis and in vitro activity of (1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives.

(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

Fragment-based identification of Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high-concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure-based drug design (SBDD) approach.

Simultaneous PIV/OH-PLIF, Rayleigh thermometry/OH-PLIF and stereo PIV measurements in a low-swirl flame.

The diagnostic techniques for simultaneous velocity and relative OH distribution, simultaneous temperature and relative OH distribution, and three component velocity mapping are described. The data extracted from the measurements include statistical moments for inflow fluid dynamics, temperature, conditional velocities, and scalar flux. The work is a first step in the development of a detailed large eddy simulation (LES) validation database for a turbulent, premixed flame. The low-swirl burner used in this investigation has many of the necessary attributes for LES model validation, including a simplified interior geometry; it operates well into the thin reaction zone for turbulent premixed flames, and flame stabilization is based entirely on the flow field and not on hardware or pilot flames.

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression.

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.

Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway.

Lysophosphatidic acid (LPA) is present at high concentrations in ascites and plasma of ovarian cancer patients. Studies conducted in experimental models demonstrate that LPA promotes ovarian cancer invasion/metastasis by up-regulating protease expression, elevating protease activity, and enhancing angiogenic factor expression. In this study, we investigated the effect of LPA on ovarian cancer migration, an essential component of cancer cell invasion. LPA stimulates both chemotaxis and chemokinesis of ovarian cancer cells and LPA-stimulated cell migration is G(I) dependent. Moreover, constitutively active H-Ras enhances ovarian cancer cell migration, whereas dominant negative H-Ras blocks LPA-stimulated cell migration, suggesting that Ras works downstream of G(i) to mediate LPA-stimulated cell migration. Interestingly, H-Ras mutants that specifically activate Raf-1, Ral-GDS, or phosphatidylinositol 3'-kinase are unable to significantly enhance ovarian cancer cell migration, suggesting that a Ras downstream effector distinct from Raf-1, Ral-GDS, and phosphatidylinositol 3'-kinase is responsible for LPA-stimulated cell migration. In this article, we demonstrate that LPA activates mitogen-activated protein kinase kinase 1 (MEKK1) in a G(i)-Ras-dependent manner and that MEKK1 activity is essential for LPA-stimulated ovarian cancer cell migration. Inhibitors that block MEKK1 downstream pathways, including MEK1/2, MKK4/7, and nuclear factor-kappa B pathways, do not significantly alter LPA-stimulated cell migration. Instead, LPA induces the redistribution of focal adhesion kinase to focal contact regions of the cytoplasm membrane, and this event is abolished by pertussis toxin, dominant negative H-Ras, or dominant negative MEKK1. Our studies thus suggest that the G(i)-Ras-MEKK1 signaling pathway mediates LPA-stimulated ovarian cancer cell migration by facilitating focal adhesion kinase redistribution to focal contacts.