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G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs (1), the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human [β.sub.2] adrenergic receptor ([β.sub.2]AR) as a guide, we designed a [β.sub.2]AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent [β.sub.2]AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound [β.sub.2]AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method (2), and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibodystabilized active structure (companion paper (3)) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonistbound active conformation spontaneously relaxes to an inactivelike conformation in the absence of a G protein or stabilizing antibody., The relationship between agonist binding to GPCRs and the conformational changes that facilitate G protein binding and activation remains largely unknown. Characterization of GPCR activation at a molecular level has [...]

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody. [ABSTRACT FROM AUTHOR]

The bacterial enzyme UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), catalyzing the first committed step of lipid A biosynthesis, represents a promising target in the development of novel antibiotics against Gram-negative bacteria. Structure, catalytic reaction mechanism and regulation of the Zn2+-dependent metalloamidase have been intensively investigated. The enzyme is required for growth and viability of Gram-negative bacteria, displays no sequence homology with any mammalian protein, but is highly conserved in Gram-negative bacteria, thus permitting the development of Gram-negative selective antibacterial agents with limited off-target effects. Several smallmolecule LpxC inhibitors have been developed, like the substrate analog TU-514 (12a), the aryloxazoline L-161,240 (13w), the sulfonamide BB-78485 (23a), the N-aroyl-L-threonine derivative CHIR-090 (24a), the sulfone-containing pyridone LpxC-3 (43e), and the uridine-based inhibitor 1-68A (47a), displaying diverse inhibitory and antibacterial activities. Most of these compounds share a Zn2+-binding hydroxamate moiety attached to a structural element addressing the hydrophobic tunnel or the UDP binding site. The butadiynyl derivative ACHN-975 (28) is the first LpxC inhibitor entering clinical trials.

A series of 6,8-diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, the affinity toward sigma(1) and sigma(2) receptors was investigated, and the growth inhibition of six human tumor cell lines was determined. The enantiopure bicyclic ketones 5a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane-2,7,9-trione) and 5b ((+)-(1S,5S)-6-allyl-8-(2,4-dimethoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane-2,7,9-trione) as well as their enantiomers ent-5a and ent-5b served as chiral building blocks, which were derived from (S)- and (R)-glutamate, respectively. Structure-affinity relationships revealed that 11a (K(i) = 154 nM), ent-11a (K(i) = 91 nM), and ent-17a (K(i) = 104 nM) are the most potent sigma(1) ligands. High sigma(2) affinity was achieved with 17b (K(i) = 159 nM) and 8b (K(i) = 400 nM). The bicyclic sigma ligands showed a selective growth inhibition of the small cell lung cancer cell line A-427 with the benzyl ethers 11 and the benzylidene derivatives 17 being the most potent compounds. 11a has a cytotoxic potency (IC(50) = 0.92 muM), which exceeds the activity of cisplatin and interacts considerably with both sigma(1) and sigma(2) receptors.