Multifaceted Approach to Determine the Antagonist Molecular Mechanism and Interaction of Ibodutant ([1-(2-Phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) at the Human Tachykinin NK₂ Receptor

Abstract

Ibodutant (MEN15596, [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK 2 R, ibodutant potency measured toward concentration-response curves to neurokinin A as pK B was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [ 3 H]nepadutant and [ 3 H]saredutant binding sites. Determined K on and K off values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK 2 Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK 2 R complex is proposed.G protein-coupled receptors (GPCRs) represent approximately 50% of current therapeutic targets. Antagonists for GPCR are so defined because of their capability to interfere with the ability of agonists in producing a given pharmacological response. The determination of the mechanism of action of an antagonist is one of the major pharmacological endeavors in drug discovery and can be helpful to predict the antagonist properties in the therapeutic situation.The tachykinin NK 2 receptor (NK 2 R) is a seven-transmembrane class A (rhodopsin-like) GPCR, which is preferentially activated by neurokinin A (NKA; HKTDSFVGLM-NH 2 ), leading to G q/11 coupling after activation of phospholipase C, thus producing inositol 3-phosphate and diacylglycerol (Almeida et al., 2004).

Gender‐related differential effect of tachykinin NK2 receptor‐mediated visceral hyperalgesia in guinea pig colon

“…Ibodutant is a non‐peptidic compound, exhibiting high‐potency and long‐lasting selective antagonist activity at the human tachykinin NK 2 receptor, both recombinant and natively expressed in the colon (Meini et al, ; Santicioli et al, ). It is effective in inhibiting colonic contractions induced by NK 2 receptor agonist stimulation in the anaesthesized guinea pig (Cialdai et al, ).…”

Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach

“…Therefore, several studies have used the competition association assay described by Motulsky and Mahan 8 to estimate the kinetic rate constants of unlabeled compounds. 9–14 In this approach, an unlabeled compound is added simultaneously with a kinetically characterized radioligand to the target protein, and binding of the radioligand is measured over time. If the competitor dissociates from the target slower than the radioligand, the radioligand association binding decreases time dependently until equilibrium is reached after an initial “overshoot.” In contrast, when the competitor dissociates faster, radioligand association binding is delayed in a monophasic manner.…”