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The vasopressin V 2 receptor (V 2 R) is a validated therapeutic target for autosomal dominant polycystic kidney disease (ADPKD), with tolvaptan being the first FDA-approved antagonist. Herein, we used Gaussian accelerated molecular dynamics simulations to investigate the spontaneous binding of tolvaptan to both active and inactive V 2 R conformations at the atomic-level. Overall, the binding process consists of two stages. Tolvaptan binds initially to extracellular loops 2 and 3 (ECL2/3) before overcoming an energy barrier to enter the pocket. Our simulations result highlighted key residues (e.g., R181, Y205, F287, F178) involved in this process, which were experimentally confirmed by site-directed mutagenesis. This work provides structural insights into tolvaptan-V 2 R interactions, potentially aiding the design of novel antagonists for V 2 R and other G protein-coupled receptors., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Racemic tolvaptan possessing an asymmetric carbon is metabolized to three pairs of monohydroxylate enantiomers of diol form with V 2 receptor antagonistic activity via CYP3A. This study aimed to develop a simultaneous quantitative liquid chromatography-tandem mass spectrometry method for 5R- and 5S-tolvaptan and their monohydroxylate enantiomers in human plasma and to apply it to patient samples. Deproteinized plasma specimens were separated using a polysaccharide derivative chiral column in a reversed-phase elution mode. The mass spectrometer was run in positive ion electrospray ionization mode. The chromatographic peaks of tolvaptan monohydroxylate enantiomers were identified by the recombinant CYP3A4/5 digestion of 5R- and 5S-tolvaptan. The calibration curves ranged over the plasma concentrations of 0.25-125 ng/mL for 5R- and 5S-tolvaptan, 0.025-12.5 ng/mL for 4R5R- and 4S5S-diols, and 0.025-38.15 ng/mL for 4S5R-, 4R5S-, 3S5R-, and 3R5S-diols with a large variation. Their pre-treatment recovery rates and matrix factors in human plasma were 85.2-112.9 % and 86.9-113.1 %, respectively. The intra- and inter-day accuracy and imprecision were 92.3-113.8 % and 3.5-14.6 % for all analytes, respectively. The plasma concentration ranges of 5R- and 5S-tolvaptan, 4R5R-, 4S5S-, 4S5R-, 4R5S-, 3S5R-, and 3R5S-diols in heart failure patients with a 5-fold dilution procedure were 0.634-28.4, 2.37-131, 0.525-15.4, 0.0970-4.08, 6.82-108, 0.271-6.49, 0.394-4.18, and 4.81-39.8 ng/mL, respectively. In conclusion, the present method has an acceptable analytical performance level and can be helpful for characterization of the plasma 5R- and 5S-tolvaptan and their monohydroxylate enantiomers in heart failure patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Purpose: We investigated the inhibitory effect and associated molecular mechanisms of tolvaptan on angiotensin II (AngII)-induced aldosterone production in vitro and in vivo., Experimental Approach: In vitro, H295R human adrenocarcinoma cells were incubated with 1 μmol·L -1 arginine vasopressin (AVP) or dDAVP, or tolvaptan (0.1, 1, and 3 μmol·L -1 ) in the presence and absence of 100 nmol·L -1 of AngII. In vivo, Sprague-Dawley rats were treated with tolvaptan 0.05% in the diet for 6 days in the presence and absence of 200 pmol·min -1 AngII., Key Results: Tolvaptan suppressed AngII-induced aldosterone production in a dose-dependent manner in H295R cells, whereas neither AVP nor dDAVP in the presence or absence of AngII altered aldosterone production, suggesting the vasopressin V 2 receptor was not involved in the inhibitory effect of tolvaptan on aldosterone synthesis. In addition, tolvaptan inhibited the AngII-induced increase in aldosterone synthase (CYP11B2) protein levels without suppressing CYP11B2 mRNA expression. Notably, tolvaptan increased the levels of unfolded protein response (UPR) marker DDIT3 and eIF2α phosphorylation (a UPR-induced event), which could block the translation of CYP11B2 mRNA into protein and thereby inhibit aldosterone production. In vivo, tolvaptan significantly inhibited AngII-induced increases in serum and adrenal aldosterone levels and CYP11B2 protein levels. This anti-aldosterone effect was associated with a reduction in the elevated systolic and diastolic BP., Conclusions and Implications: Tolvaptan inhibited AngII-stimulated aldosterone production via a V 2 receptor-independent pathway, which can counteract or even surpass its potential activating effect of diuresis-induced aldosterone secretion in certain aldosterone-mediated pathological conditions., (© 2019 The British Pharmacological Society.)