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Luteolin, a flavonoid widely distributed in the plant kingdom, contains two benzene rings and hydroxyl groups, and this structural specificity contributes to its diverse biological activities. However, no previous studies have simultaneously investigated the therapeutic potency of luteolin isolated from a plant as an antipsychotic and antidepressant. Here, luteolin exhibited selective inhibition of hMAO-A (IC 50 = 8.57 ± 0.47 μM) over hMAO-B (IC 50 > 100 μM). In silico proteochemometric modeling predicted promising targets of luteolin, and verification via cell-based G protein-coupled receptor functional assays showed that luteolin is a selective antagonist of the vasopressin receptor V 1A R (IC 50 = 19.49 ± 6.32 μM) and the dopamine D 4 receptor (IC 50 = 39.59 ± 1.46 μM). Molecular docking showed the tight binding of luteolin with a low binding score and the high stability of the luteolin-receptor complex, corroborating its functional effect. Thus, hMAO-A, hD 4 R, and hV 1A R are prime targets of luteolin and potential alternatives for the management of neurodegenerative diseases.

Hydrogen-deuterium exchange-mass spectrometry (HDXMS) is a powerful technology to characterize conformations and conformational dynamics of proteins and protein complexes. HDXMS has been widely used in the field of therapeutics for the development of protein drugs. Although sufficient sequence coverage is critical to the success of HDXMS, it is sometimes difficult to achieve. In this study, we developed a HDXMS data analysis strategy that includes parallel post-translational modification (PTM) scanning in HDXMS analysis. Using a membrane-delimited G protein-coupled receptor (vasopressin type 2 receptor; V 2 R) and a cytosolic protein (Na + /H + exchanger regulatory factor-1; NHERF1) as examples, we demonstrate that this strategy substantially improves protein sequence coverage, especially in key structural regions likely including PTMs themselves that play important roles in protein conformational dynamics and function.

The anti and syn isomers of tolvaptan-type compounds, N-benzoyl-5-hydroxy-1-benzazepines (5a-c), were prepared in a stereocontrolled manner by biasing the conformation with a methyl group at C9 and C6, respectively, and the enantiomeric forms were separated. Examination of the affinity at the human vasopressin receptors revealed that the axial chirality (aS) plays a more important role than the central chirality at C5 in receptor recognition, and the most preferable form was shown to be (E,aS,5S).

From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.

Protein misfolding is a process in which proteins are unable to attain or maintain their biologically active conformation. Factors contributing to protein misfolding include missense mutations and intracellular factors such as pH changes, oxidative stress, or metal ions. Protein misfolding is linked to a large number of diseases such as cystic fibrosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and less familiar diseases such as Gaucher's disease, nephrogenic diabetes insipidus, and Creutzfeldt-Jakob disease. In this Perspective, we report on small molecules that bind to and stabilize the aberrant protein, thereby helping it to attain a native or near-native conformation and restoring its function. The following targets will be specifically discussed: transthyretin, p53, superoxide dismutase 1, lysozyme, serum amyloid A, prions, vasopressin receptor 2, and α-1-antitrypsin.

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.

Phosphorylation plays vital roles in the regulation and function of the V2 vasopressin receptor (V2R), a G protein-coupled receptor (GPCR) that is responsible for maintaining water homeostasis in the kidney. Through a combination of immunoaffinity purification, immobilized metal affinity chromatography, and nanoflow liquid chromatography tandem mass spectrometry, we identified a novel phosphorylation site (Ser(255)) in the third intracellular loop of human V2R. We showed that the third intracellular loop could be phosphorylated in vitro by protein kinase A, but not by Akt kinase, although sequence motif analysis predicated otherwise. The analytical procedures and methodologies described in this study should be generally applicable for identifying the endogenous phosphorylation sites in other GPCRs, overcoming the limitations of conventional approaches such as sequence motif analysis and site-directed mutagenesis.

In this study, four cyclic vasopressin (CYFQNCPRG-NH(2), AVP) analogues substituted at positions 2 and 3 with four combinations of enantiomers of N-methylphenylalanine have been investigated. Three-dimensional structures of analogues have been formerly determined using NMR spectroscopy in dimethyl sulfoxide. Three-dimensional models of the vasopressin and oxytocin receptors were constructed by combining the multiple sequence alignment and the RD crystal structure as a template. The analogues have been docked into the receptor using the AutoDock program. The relaxation of the receptor-ligand complexes using energy minimization, followed by the constrained simulated annealing protocols (CSA), has been performed. The receptor-bound conformations of the investigated analogues have been proposed. We concluded that the N-methylated residues at positions 2 and 3 act as a structural restraint, determining the conformation of analogues, their location inside the receptor cavity, and mutual arrangement of the aromatic side chains. The conserved polar residues constitute the handles keeping the biologically active analogues inside the binding cavity. The Arg(8)-D(2.65) salt bridge might be responsible for analogue-selective binding in OTR and V1aR versus V2R, where the positively charged K(2.65) 100 is present at the equivalent position.

The seven-transmembrane-spanning G protein-coupled receptor (GPCR) superfamily plays many important roles in basic biology, human health, and human disease. Here, well-resolved solution NMR spectra are presented for a human GPCR, the vasopressin V2 receptor in detergent micelles. The quality of the NMR spectra indicates that backbone resonance assignments for a majority of resonances are feasible. The key to obtaining high quality spectra appears to be the coupling of methods for expressing the receptor into membranes rather than into inclusion bodies, with use of a biochemically mild lysolipid detergent for membrane extraction, protein purification, and NMR sample preparation.

Oxytocin receptor (OTR) activates the GTP-binding protein Galpha(q). To investigate whether the N-terminal region of the fourth intracellular domain of this receptor, which forms putative helix 8, plays a role in coupling, its hydrophilic residues (H7.59, H7.62, E7.63, Q7.66, and R7.67) were mutated individually to alanine. In COSM6 cells, these mutants were expressed at equivalent concentrations, but at lower concentrations than OTR. Alanine substitution for H7.62 or Q7.66 did not substantially affect the affinity for OT (K(d) = 0.63 and 0.48 nM, respectively, vs 0.52 nM for the wild type), whereas substitution for H7.59, E7.63, or R7.67 reduced the affinity 5-6-fold. When expressed at equal concentrations, OTR-H7.62/A and OTR-Q7.66/A stimulated phosphatidylinositide turnover as well as OTR, whereas OTR-H7.59/A, OTR-E7.63/A, and OTR-R7.67/A exhibited an impaired ability to respond to OT. Therefore, residues H7.59, E7.63, and R7.67 within the putative hydrophilic interface appeared to influence both the OTR conformation and Galpha(q) coupling. To explore this further, five multiple alanine substitution mutants were constructed. Alanine modification at H7.62 and Q7.66 did not substantially affect the affinity for OT (K(d) = 0.75 nM), whereas any combination of alanine substitutions for H7.59, E7.63, and R7.67 produced mutant receptors that lost high-affinity ligand binding. While OTR-(H7.62,Q7.66)/A exhibited PLC activation equivalent to that of OTR, receptors with two or more changes in H7.59, E7.63, and R7.67 lost the ability to respond to OT in a dose-dependent manner. Five residues (L7.60, F7.61, L7.64, V7.65, and F7.68) in the opposite hydrophobic interface were also mutated to alanine. None of these substitutions affected ligand binding; only OTR-(L7.60,F7.61)/A had a somewhat weaker ability to activate PLC. These data are consistent with the prediction that these residues lie within an amphipathic alpha-helix and emphasize the importance of this hydrophilic interface, and particularly of H7.59, E7.63, and R7.67, in OTR function.

Vasopressin (VP) receptors belong to the widespread G protein-coupled receptor family. The crucial role of VP receptor intracellular loops in the coupling with the heterotrimeric G proteins was previously demonstrated by construction of a vasopressin receptor chimera. Yet, no fine structural data are available concerning the receptor molecular determinants involved in their interactions with G proteins. In this study, we synthesized both a linear and a cyclic form of the second intracellular loop (i2) of the human V(1a) vasopressin receptor isoform that is important for the interaction between the alphaq/alpha11 G protein and the receptor. These two peptides are biologically active. They specifically inhibit vasopressin binding to the V(1a) receptor, suggesting that the corresponding endogenous peptides contribute to the structure of the vasopressin binding site via intra- or intermolecular interactions with the core of the V(1a) receptor. The i2 peptide structures were determined by (1)H NMR. Both exhibit a helix and helical elements in their N- and C-terminal parts, respectively, separated by a turn imposed by a proline residue. More interestingly, the central Pro-Leu motif conserved in many GPCRs and thought to be important for coupling to G proteins can adopt different conformations. The "U" shape structure of the i2 loop is compatible with its anchoring to transmembrane domains III and IV and is very similar to the shape of bovine rhodopsin i2. Altogether, these data contribute to a better understanding of the structure of a not yet crystallized GPCR using the mimetic peptide approach.