Tolvaptan (OPC-41061) is an orally bioavailable, selective, arginine vasopressin receptor 2 (V2, AVPR2) antagonist that can be used to treat hyponatremia.

V2 (human):1.9 nM, NTCP:31.6 μM, MRP2:> 50 μM, HepG2 cells:27.1 µM, V1 (human):4.9 nM (Ki), V1 (human):25 nM, BSEP:∼41.5 μM, V2 (human):0.85 nM (Ki), H69 cells:53.6 μM, V2 receptor:3 nM

Cell-free autophosphorylation assay using time-resolved fluorometry: Neratinib is prepared as 10 mg/mL stocks in DMSO and diluted in 25 mM HEPES (pH 7.5; 0.002 ng/mL-20 μg/mL). Purified recombinant COOH-terminal fragments of HER2 (amino acids 676-1255) or epidermal growth factor receptor (EGFR) (amino acids 645-1186) [diluted in 100 mM HEPES (pH 7.5) and 50% glycerol] is incubated with increasing concentrations of Neratinib in 4 mM HEPES (pH 7.5), 0.4 mM MnCl2, 20 μM sodium vanadate, and 0.2 mM DTT for 15 minutes at room temperature in 96-well ELISA plates. The kinase reaction is initiated by the addition of 40 μM ATP and 20 mM MgCl2 and allowed to proceed for 1 hour at room temperature. Plates are washed, and phosphorylation is detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well). After washing and enhancement steps, signal is detected using a Victor2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615 nm). The concentration of Neratinib that inhibits receptor phosphorylation by 50% (IC50) is calculated from inhibition curves.