Role of selective alpha and beta adrenergic receptor mechanisms in rat jejunal longitudinal muscle contractility

Gut motility is modulated by adrenergic mechanisms. The aim of our study was to examine mechanisms of selective adrenergic receptors in rat jejunum. Spontaneous contractile activity of longitudinal muscle strips from rat jejunum was measured in 5-ml tissue chambers. Dose-responses (six doses, 10(-7) -3 x 10(-5)M) to norepinephrine (NE, nonspecific), phenylephrine (PH, alpha1), clonidine (C, alpha2), prenalterol (PR, beta1), ritodrine (RI, beta2), and ZD7714 (ZD, beta3) were evaluated with and without tetrodotoxin (TTX, nerve blocker). NE(3 x 10(-5)M) inhibited 74 +/- 5% (mean +/- SEM) of spontaneous activity. This was the maximum effect. The same dose of RI(beta2), PH(alpha1), or ZD(beta(3)) resulted in an inhibition of only 56 +/- 5, 43 +/- 4, 33 +/- 6, respectively. The calculated concentration to induce 50% inhibition (EC50) of ZD(beta3) was similar to NE, whereas higher concentrations of PH(alpha1) or RI(beta2) were required. C(alpha2) and PR(beta1) had no effect. TTX changed exclusively the EC50 of RI from 4.4 +/- 0.2 to 2.7 +/- 0.8% (p < 0.04). Contractility was inhibited by NE (nonspecific). PH(alpha1), RI(beta2), and ZD(beta3) mimic the effect of NE. TTX reduced the inhibition by RI. Our results suggest that muscular alpha1, beta2, and beta3 receptor mechanisms mediate adrenergic inhibition of contractility in rat jejunum. beta2 mechanisms seem to involve also neural pathways.

Conformationally homogeneous heterocyclic pseudotetrapeptides as three-dimensional scaffolds for rational drug design: receptor-selective somatostatin analogues

A would-be amide: A 1,4-disubstituted 1,2,3-triazole was used as a surrogate for a trans amide bond to create a library of 16 diastereomeric pseudotetrapeptides as beta-turn mimetics. High-resolution structural analysis indicated that these scaffolds adopt distinct, rigid, conformationally homogeneous beta-turn-like structures (see example), some of which bind somatostatin receptor subtypes selectively, and some of which show broad-spectrum activity.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the CNS and PNS that is activated by the endogenous agonist serotonin (5-hydroxytryptamine, 5-HT). 5-HT3R is the only serotonin receptor belonging to the Cys-loop superfamily of neurotransmitter receptors. Different structural biology approaches can be applied, such as crystallization and x-ray analysis. Nonetheless, characterizing the exact ligand binding site(s) of these dynamic receptors is still challenging. The use of photo-crosslinking probes is an alternative validated approach allowing identification of regions in the protein that are important for the binding of small molecules. We designed our probes based on the core structure of the 5-HT3R antagonist granisetron, a FDA approved drug used for the treatment of chemotherapy-induced nausea and vomiting. We synthesized a small library of photo-crosslinking probes by conjugating diazirines and benzophenones via various linkers to granisetron. We were able to obtain several compounds with diverse linker lengths and different photo-crosslinking moieties that show nanomolar binding affinity for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Several experiments showed unambiguously that we are able to photo-crosslink our probes with the receptor site-specifically. The functionalised protein was analysed by Western blot and MS-analysis. This yielded the exact covalent modification site, corroborating current ligand binding models derived from mutagenesis and docking studies.

Glucagonlike peptide-1 receptor: an example of translational research in insulinomas: a review

Glucagonlike peptide-1 receptors (GLP-1R) play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Exendin-4 is a GLP-1 analog that has a longer half-life than GLP-1. Targeting GLP-1R by (111)In-DOTA-exendin-4 or (111)In-DPTA-exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new noninvasive technique has the potential to replace the invasive localization by selective arterial stimulation and venous sampling.

Icatibant, a new bradykinin-receptor antagonist, in hereditary angioedema

Hereditary angioedema is characterized by recurrent attacks of angioedema of the skin, larynx, and gastrointestinal tract. Bradykinin is the key mediator of symptoms. Icatibant is a selective bradykinin B2 receptor antagonist.