Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

Role of endothelin ETA receptors in the hypertension induced by the VEGFR-2 kinase inhibitors axitinib and lenvatinib in conscious freely-moving rats.

Receptor tyrosine kinase inhibitors (RTKIs) suppress tumour growth by targeting vascular endothelial growth factor receptor 2 (VEGFR-2) which is an important mediator of angiogenesis. Here, we demonstrate that two potent RTKIs, axitinib and lenvatinib, are associated with hypertensive side effects. Doppler flowmetry was used to evaluate regional haemodynamic profiles of axitinib and lenvatinib. Male Sprague Dawley rats (350-500 g) were instrumented with Doppler flow probes (renal and mesenteric arteries and descending abdominal aorta) and catheters (jugular vein and distal abdominal aorta, via the caudal artery). Rats were dosed daily with axitinib (3 or 6 mg.kg-1) or lenvatinib (1 or 3 mg.kg-1) and regional haemodynamics were recorded over a maximum of 4 days. Both RTKIs caused significant (p < 0.05) increases in mean arterial pressure (MAP), which was accompanied by significant (p < 0.05) vasoconstriction in both the mesenteric and hindquarters vascular beds. To gain insight into the involvement of endothelin-1 (ET-1) in RTKI-mediated hypertension, we also monitored heart rate (HR) and MAP in response to axitinib or lenvatinib in animals treated with the ETA receptor selective antagonist sitaxentan (5 mg.kg-1) or the mixed ETA/ETB receptor antagonist bosentan (15 mg.kg-1) over two days. Co-treatment with bosentan or sitaxentan markedly reduced the MAP effects mediated by both RTKIs (p < 0.05). Bosentan, but not sitaxentan, also attenuated ET-1 mediated increases in HR. These data suggest that selective antagonists of ETA receptors may be appropriate to alleviate the hypertensive effects of axitinib and lenvatinib.

Heterodimeric Analogues of the Potent Y1R Antagonist 1229U91, Lacking One of the Pharmacophoric C-Terminal Structures, Retain Potent Y1R Affinity and Show Improved Selectivity over Y4R.

The cyclic dimeric peptide 1229U91 (GR231118) has an unusual structure and displays potent, insurmountable antagonism of the Y1 receptor. To probe the structural basis for this activity, we have prepared ring size variants and heterodimeric compounds, identifying the specific residues underpinning the mechanism of 1229U91 binding. The homodimeric structure was shown to be dispensible, with analogues lacking key pharmacophoric residues in one dimer arm retaining high antagonist affinity. Compounds 11d-h also showed enhanced Y1R selectivity over Y4R compared to 1229U91.

Optically Pure, Structural, and Fluorescent Analogues of a Dimeric Y4 Receptor Agonist Derived by an Olefin Metathesis Approach.

The dimeric peptide 1 (BVD-74D, as a diastereomeric mixture) is a potent and selective neuropeptide Y Y4 receptor agonist. It represents a valuable candidate in developing traceable ligands for pharmacological studies of Y4 receptors and as a lead compound for antiobesity drugs. Its optically pure stereoisomers along with analogues and fluorescently labeled variants were prepared by exploiting alkene metathesis reactions. The (2R,7R)-diaminosuberoyl containing peptide, (R,R)-1, had markedly higher affinity and agonist efficacy than its (S,S)-counterpart. Furthermore, the sulfo-Cy5 labeled (R,R)-14 retained high agonist potency as a novel fluorescent ligand for imaging Y4 receptors.

Synthetic routes to the Neuropeptide Y Y1 receptor antagonist 1229U91 and related analogues for SAR studies and cell-based imaging.

The potent Y1 receptor antagonist, 1229U91 has an unusual cyclic dimer structure that makes syntheses of analogue series quite challenging. We have examined three new routes to the synthesis of such peptides that has given access to novel structural variants including heterodimeric compounds, ring size variants and labelled conjugates. These compounds, including a fluorescently labelled analogue VIII show potent antagonism that can be utilised in studying Y1 receptor pharmacology.

The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the ß2-adrenergic receptor.

Recent high resolution x-ray structures of the ß2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.

Thyroid-stimulating monoclonal antibodies.

Thyrotropin (TSH) receptor monoclonal antibodies (TSHR mAbs) were obtained from cDNA-immunized NMRI mice. Three mAb immunoglobulin Gs (IgGs) (TSmAbs 1-3) that had distinct V(H )and V(L) region sequences stimulated cyclic adenosine monophosphate (cAMP) production in isolated porcine thyroid cells greater than 10x basal and as little as 20 ng/mL (0.13 nmol/L) of TSmAb 1 IgG caused a 2x basal stimulation. TSmAb 1 and 2 Fab fragments were also effective stimulators and thyroid-stimulating activities of the IgGs and Fabs were confirmed using TSHR transfected Chinese hamster ovary (CHO) cells. The TSmAbs also inhibited (125)I-labeled TSH binding to TSHR-coated tubes by 50% or more at concentrations of 1 microg/mL or less and gave 15%-20% inhibition at 20-50 ng/mL. (125)I-labeled TSmAbs bound to TSHR-coated tubes with high affinity (approximately 10(10) L/mol) and this binding was inhibited by TSHR autoantibodies with both TSH agonist and antagonist activities. Inhibition of labeled TSmAb binding by Graves' sera correlated well with inhibition of TSH binding (r = 0.96; n = 18; p < 0.001 for TSmAb 2). The TSmAbs have considerable potential as (1) new probes for TSHR structure-function studies, (2) reagents for new assays for TSHR autoantibodies, and (3) alternatives to recombinant TSH in various in vivo applications.