Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Alterations in the steroid hormone receptor co-chaperone FKBPL are associated with male infertility: a case-control study

Background: Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients.

Identification of Determinants of Glucose-Dependent Insulinotropic Polypeptide Receptor That Interact with N-Terminal Biologically Active Region of the Natural Ligand

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.

Two Arginine-Glutamate Ionic Locks Near the Extracellular Surface of FFAR1 Gate Receptor Activation

Activation of a number of class A G protein-coupled receptors (GPCRs) is thought to involve two molecular switches, a rotamer toggle switch within the transmembrane domain and an ionic lock at the cytoplasmic surface of the receptor; however, the mechanism by which agonist binding changes these molecular interactions is not understood. Importantly, 80% of GPCRs including free fatty acid receptor 1 (FFAR1) lack the complement of amino acid residues implicated in either or both of these two switches; the mechanism of activation of these GPCRs is therefore less clear. By homology modeling, we identified two Glu residues (Glu-145 and Glu-172) in the second extracellular loop of FFAR1 that form putative interactions individually with two transmembrane Arg residues (Arg-183(5.39) and Arg-258(7.35)) to create two ionic locks. Molecular dynamics simulations showed that binding of agonists to FFAR1 leads to breakage of these Glu-Arg interactions. In mutagenesis experiments, breakage of these two putative interactions by substituting Ala for Glu-145 and Glu-172 caused constitutive receptor activation. Our results therefore reveal a molecular switch for receptor activation present on the extracellular surface of FFAR1 that is broken by agonist binding. Similar ionic locks between the transmembrane domains and the extracellular loops may constitute a mechanism common to other class A GPCRs also.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active G alpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q-)dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

Discovery of novel Agonists and antagonists of the free fatty acid receptor 1 (FFAR1) using virtual screening

The G-protein-coupled receptor free fatty acid receptor 1 (FFAR1), previously named GPR40, is a possible novel target for the treatment of type 2 diabetes. In an attempt to identify new ligands for this receptor, we performed virtual screening (VS) based on two-dimensional (2D) similarity, three-dimensional (3D) pharmacophore searches, and docking studies by using the structure of known agonists and our model of the ligand binding site, which was validated by mutagenesis. VS of a database of 2.6 million compounds followed by extraction of structural neighbors of functionally confirmed hits resulted in identification of 15 compounds active at FFAR1 either as full agonists, partial agonists, or pure antagonists. Site-directed mutagenesis and docking studies revealed different patterns of ligand-receptor interactions and provided important information on the role of specific amino acids in binding and activation of FFAR1.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors.