Identification of antagonists to the vasotocin receptor sub-type 4 (VT4R) involved in stress by molecular modelling and verification using anterior pituitary cells

Abstract

The vasotocin receptor family is homologous to the mammalian vasopressin G-protein coupled receptor (GPCR) family. The vasotocin receptor 2 (VT2R) and 4 (VT4R) have recently been shown to play important role(s) in the neuroendocrine regulation of stress in birds. A homology-based structural model of VT4R of the domestic chicken, Gallus gallus, was built using the sophisticated SYBYL-X suite. The structure of VT4R built with and without extra- and intracellular unstructured loops showed a seven-helix transmembrane domain, which is a characteristic feature of GPCRs. Several agonists and antagonists were screened by molecular docking to map their potential binding sites on the structure of VT4R. Interestingly, the presence of the N-terminal, intracellular and extracellular loops and C-terminal amino acid sequences emerging from the transmembrane domains during molecular docking appeared to influence the binding interface of the peptide agonists and peptide/non-peptide antagonists on the VT4R. The presence of unstructured loops, however, did not affect the relative binding affinity ranking of the peptide antagonists to VT4R. In general, the natural ligand, arginine vasotocin and the peptide/non-peptide antagonists were observed to be more deeply buried in the receptor. Results of in vitro inhibition experiments, using cultured anterior pituitary cells, showed excellent agreement with the binding affinity of the antagonists predicted by molecular docking. The results of this study provide valuable clues for the rational design of novel pharmaceutical compounds capable of blocking or attenuating the stress response.

Central effect of vasotocin 4 receptor (VT4R/V1aR) antagonists on the stress response and food intake in chicks given neuropeptide Y (NPY)

“…Their conclusion was that the arginine vasopressin V1aR signal acts as an inhibitory system to suppress the NPY orexigenic action via the central nervous system and that blockade of the V1aR lead to increased food intake due to enhanced NPY sensitivity [23]. Our data utilizing an effective antagonist for the mammalian V1aR, SR-49059 [30] has shown it to be efficacious for blocking the chicken VT4R in vitro [11] and in vivo results of the current study. Due to the significant decrease in plasma CORT when SR-49059 was administered prior to immobilization compared to stressed birds given saline ICV, and the increased CORT plasma levels in birds given NPY compared to saline or SR-49059 administered control groups suggest that SR-49059 effectively blocked the VT4R and attenuated its activation due to inhibition of the binding of neuropeptides released by the stress procedure immobilization or central injection of the peptide NPY.…”

“…Data obtained in broiler chicks, gallus gallus, four to five weeks of age, have shown that administration of antagonists of the mammalian vasopressin V1aR have likewise been effective in blocking the avian VT4R that has been proposed to be equivalent to the mammalian V1aR based upon its sequence homology [28], Genbank ACCN ABV24997) and key amino acid residues at specific sites that confer specificity to vasopressin-like residues [8,29]. Indeed Jayanthi et al (2014) showed that molecular models of both the Manning compound and SR-49059 were effective in displaying effective binding values in an analysis utilizing a 3D computer-based model built for the avian VT4R as well as an in vitro assay comprising chicken pituitary cells where the same two receptor blockers inhibited the expression of POMC hnRNA.…”

“…Antagonists used in this study were H-5350, Manning compound (Bachem, Torrance, CA) and SR-49059 (Sigma-Aldrich, St. Louis, MO). The compounds and doses were selected based upon results of a previous in vitro cell culture study [11] and a preliminary ICV injection study.…”

“…Of the two receptors (VT2R and VT4R), the VT4R has been shown located not only in the anterior pituitary [5] but also in the brain of chickens [7] as well as the brains of the white-throated sparrow and zebra finch [8]. Effective antagonists of the VT4R have been identified, specifically the Manning compound, H-5350 [9,10] and SR-49059 [11]. The most efficacious blocker of the VT4R in chickens was shown to be SR-49059, based upon its binding value utilizing the development of a 3D model of the receptor as well as an in vitro blocking assay using a primary avian pituitary cell culture method [11].…”

“…Effective antagonists of the VT4R have been identified, specifically the Manning compound, H-5350 [9,10] and SR-49059 [11]. The most efficacious blocker of the VT4R in chickens was shown to be SR-49059, based upon its binding value utilizing the development of a 3D model of the receptor as well as an in vitro blocking assay using a primary avian pituitary cell culture method [11]. We next planned to test the effectiveness of the identified antagonists at the organismal level to attenuate CORT release following stress as well as determine whether the avian VT4R functions as the mammalian V1aR and plays a role in the neuroendocrine regulation of food intake.…”