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Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures(1-4) and ultra-large libraries of virtual compounds(5,6). However, to keep pace with the explosive growth of virtual libraries, such as REadily AvaiLable for synthesis (REAL) combinatorial libraries(7), new approaches to compound screening are needed(8,9). Here we introduce a novel synthon-based approach, V-SYNTHES, to perform hierarchical structure-based screening of REAL library of more than 11 billion compounds. V-SYNTHES first identifies the best synthon-scaffold combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach allows rapid detection of the best-scoring compounds in the Giga-scale chemical space while performing docking of only a small fraction (

The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 A and 3.3 A, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros–Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents. Structural and functional studies show that the MT2 melatonin receptor, unlike the MT1 receptor, contains an extracellular opening for ligand entry, shedding light on receptor subtype specificity.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures

Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.

Crystal structures provide first atomic-level insights into δ-opioid receptor activation by two structurally diverse agonists., Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.

Two distinct antagonist-bound structures of CysLT1R reveal unique ligand-binding modes and signaling mechanisms., The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms

Tryptophan indole 15N–1H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15N–1H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15N–1H resonances is challenging. Here we exploit the unique indole 15N–1H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A2AAR global protein fold and ligand binding activity are preserved in these A2AAR variants. The indole 15N–1H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15N–1H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the GαS intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.

Angiotensin II type 1 receptor (AT(1)R) is the primary blood pressure regulator. AT(1)R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT(1)R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT(1)R in complex with an inverse agonist olmesartan (Benicar (TM)), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-35(1.39), Trp-84(2.60), and Arg-167(ECL2), similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT(1)R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N111(3.35)A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the beta-arrestin-biased peptide TRV120027.

Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-A resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.

Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor (A2AAR), D522.50N and S913.39A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G protein signaling. Utilizing the D2.50N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery.

SummaryUnderstanding the mechanism by which ligands affect receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs), we currently pursue a brute-force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand's ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair.

Background. Oxytocics are routinely used in an attempt to prevent excessive blood loss during cesarean section. Misoprostol, a potent uterotonic agent, has been reported to be useful in the prevention and treatment of postpartum hemorrhage by several investigators but its use during cesarean section has not been described. The objective of this study was to randomly compare the effectiveness of oral misoprostol with intravenous syntocinon on blood loss during elective cesarean sections under regional anesthesia.Methods. Sixty pregnant women were randomized either to receive misoprostol 400 micrograms orally or syntocinon 10 IU intravenously during cesarean section. The primary outcome measure was intra-operative blood loss as estimated by physicians, and by values of preoperative and postoperative hemoglobin concentration and hematocrit. Demographic characteristics of the subjects and outcomes were compared using c-square test for categorical and two-sample t-test for continuous data.Results. Baseline cha...

The crystal structure of the human kappa opioid receptor (hKOR) in complex with antagonist JDTic had been previously solved to 2.9 A (Wu et al, 2012, Nature 485, 327-332). From this crystal structure, key receptor-ligand interactions were observed and extended via modeling to GNTI, β-NNTA, and other hKOR ligands. However, a major hurdle still remains in elucidating hKOR in different states of activation. Having this information would lead to a more comprehensive understanding of hKOR activation and function, informing design of more efficient analgesics with reduced side effects. Specifically, we seek to utilize selective hKOR ligands to trap hKOR in conformational states that correlate to the properties of each ligand. Here we present our work in progress on the design of novel hKOR constructs and their complexes with agonists, antagonists, and biased ligands.