MLS000069819 SMR000059217 DIGOXIN US10668094, Compound Digoxin cid_2724385 BDBM46355
- Mikkaichi, T; Suzuki, T; Onogawa, T; Tanemoto, M; Mizutamari, H; Okada, M; Chaki, T; Masuda, S; Tokui, T; Eto, N; Abe, M; Satoh, F; Unno, M; Hishinuma, T; Inui, K; Ito, S; Goto, J; Abe, T Isolation and characterization of a digoxin transporter and its rat homologue expressed in the kidney. Proc Natl Acad Sci U S A 101: 3569-74 (2004)
- Katz, A; Tal, DM; Heller, D; Haviv, H; Rabah, B; Barkana, Y; Marcovich, AL; Karlish, SJ Digoxin derivatives with enhanced selectivity for the a2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits. J Biol Chem 289: 21153-62 (2014)
- Alves, SL; Paixão, N; Ferreira, LG; Santos, FR; Neves, LD; Oliveira, GC; Cortes, VF; Salomé, KS; Barison, A; Santos, FV; Cenzi, G; Varotti, FP; Oliveira, SM; Taranto, AG; Comar, M; Silva, LM; Noël, F; Quintas, LE; Barbosa, LA; Villar, JA ¿-Benzylidene digoxin derivatives synthesis and molecular modeling: Evaluation of anticancer and the Na,K-ATPase activity effect. Bioorg Med Chem 23: 4397-404 (2015)
- ChEMBL_836269 (CHEMBL2076282) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_836601 (CHEMBL2077342) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_836744 (CHEMBL2075166) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_836745 (CHEMBL2075167) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_837273 (CHEMBL2075362) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_837274 (CHEMBL2075363) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_838497 (CHEMBL2078125) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 5 uM) in Caco-2 cells
- ChEMBL_836270 (CHEMBL2076283) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 0.025 uM) in MDR1-expressing LLC-PK1 cells
- ChEMBL_837275 (CHEMBL2075364) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 0.1 uM) in MDR1-expressing LLC-PK1 cells
- ChEMBL_838474 (CHEMBL2076045) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) (Digoxin: 0.1 uM) in MDR1-expressing LLC-PK1 cells
- ChEMBL_837488 (CHEMBL2075976) TP_TRANSPORTER: inhibition of Digoxin uptake in Xenopus laevis oocytes
- ChEMBL_838982 (CHEMBL2078499) TP_TRANSPORTER: inhibition of Digoxin uptake in Xenopus laevis oocytes
- ChEMBL_838983 (CHEMBL2078500) TP_TRANSPORTER: inhibition of Digoxin uptake in Xenopus laevis oocytes
- ChEMBL_836924 (CHEMBL2075764) TP_TRANSPORTER: inhibition of Digoxin uptake in OATP4C1-expressing MDCK cells
- ChEMBL_2016318 (CHEMBL4669896) Inhibition of human ABCG2 expressed in MDCK2-LE cells in presence of digoxin
- ChEMBL_836632 (CHEMBL2077474) TP_TRANSPORTER: inhibition of Digoxin uptake in Oatp2-expressing LLC-PK1 cells
- ChEMBL_838750 (CHEMBL2078576) TP_TRANSPORTER: transepithelial transport of digoxin (basal to apical) in Caco-2 cells
- ChEMBL_838984 (CHEMBL2078501) TP_TRANSPORTER: inhibition of Digoxin uptake in Oatp2-expressing LLC-PK1 cells
- ChEMBL_838985 (CHEMBL2078502) TP_TRANSPORTER: inhibition of Digoxin uptake in Oatp2-expressing LLC-PK1 cells
- ChEMBL_730589 (CHEMBL1695310) Inhibition of human MDR1-mediated digoxin permeability expressed in pig LLC-PK1 cells
- ChEMBL_838495 (CHEMBL2076066) TP_TRANSPORTER: inhibition of Digoxin transepithelial transport (basal to apical) in MDR1-expressing MDCK cells
- ChEMBL_2287196 Inhibition of P-gp mediated efflux in human LS180 cells using digoxin as substrate incubated for 90 mins by fluorescence assay
- ChEMBL_837645 (CHEMBL2076598) TP_TRANSPORTER: inhibition of Doxorubicin transepithelial transport (basal to apical) (Digoxin: 0.8 uM) in MDR1-expressing LLC-PK1 cells
- ChEMBL_1792872 (CHEMBL4264791) Displacement of [3H]-Digoxin from recombinant human GST-fused RORgammat LBD after 1 to 4 hrs by scintillation counting method
- ChEMBL_1483315 (CHEMBL3539399) Inhibition of MDR1 (unknown origin) expressed in MDCK2 cells assessed as basolateral to apical transport of [3H]digoxin after 90 mins by liquid scintillation counting analysis
- DNA Strand-Transfer Activity Assay In the strand-transfer assay, the biotinylated donor DNA was bound to streptavidin-coated plates, and integrase was added to each well to allow processing of the donor DNA. Serially diluted compounds and DIG-tagged target DNA were then added to each well, and 3-end joining reaction was allowed to proceed for 30 min. Signal was detected using horseradishperoxidase-conjugated anti-Digoxin antibody through chemiluminescence measurement.
- Integrase 3-End Joining Assay In the 3-end joining assay, the biotinylated donor DNA was bound to streptavidin-coated plates, and integrase was added to each well to allow processing of the donor DNA. Serially diluted compounds and DIG-tagged target DNA were then added to each well, and 3-end joining reaction was allowed to proceed for 30 min. Signal was detected using horseradishperoxidase-conjugated anti-Digoxin antibody through chemiluminescence measurement.
- Selective Inhibition Assays of Isolated Na,K-ATPase To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXTD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.Na,K-ATPase activity of α/βPFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).The Na,K-ATPase activities were α1β1, 21.5±5.3 μmoles/min/mg; α2β1, 18.7±1.8 μmoles/min/mg, and α2β3, 10.7±1.9 μmoles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K0.5 for activation by K: α1β1-1.25±0.05 mM, α2β1-2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.Selectivity of the compounds for various isolated isoforms of human Na,K-ATPase was determined essentially as described before [Katz, A. et al., J Biol Chem., 2010, 285(25), pp. 19582-19592].ATPase activity assays as well as titrations with NaCl, KCl and vanadate were performed as described in Lifshitz-2007 and Loayza-1998 using PiColorLock malachite green assay (Inova Bioscience). Inhibitor assays were performed as described in Katz-2010. [3H]ouabain binding and K+-[3H]digoxin displacement assays were performed as described in Katz-2010.The percent inhibition VCG/V0 was calculated and Ki values were obtained by fitting the data to the function VCG/V0=Ki/([CG]+Ki)+c (CG stands for cardiac glycoside). Inhibition was estimated in 3-5 separate experiments and average Ki values±standard error of the mean (SEM) were calculated. The ratios Ki α1β1/α2β1, α1β1/α2β2 and α1β1/α2β3 was calculated for each compound.
- In Vitro Inhibition Activity To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXYD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.Na,K-ATPase activity of α/βFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).The Na,K-ATPase activities were α1β1, 21.5±5.3 moles/min/mg; α2β1, 18.7±1.8 moles/min/mg, and α2β3, 10.7±1.9 moles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K0.5 for activation by K: α1β1 1.25±0.05 mM, α2β1 2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.
- Caco-2 cells assay To determine potency of compounds on inhibition of transporters, bi-directional transport studies are performed in Caco-2 cells (American Type Culture Collection, Manassas, Va.) at 37° C. in air, according toXia et al. (Expression, localization and functional characteristics of breast cancer resistance protein in Caco-2 cells. Drug Met. Disp., 33 (5): 637-643 (2005)). Prior to each experiment, the confluent cell monolayers on Transwell inserts are washed and equilibrated for 30 minutes with transport media (Hank's balanced salt solution (HBSS) containing 10 mM of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) and 10 mM of glucose, pH 7.4). The experiment is initiated by adding a solution containing the 3H-digoxin (25 nM, substrate for p-gp), or 3H-E3S (17 nM, substrate for BCRP) to either the apical (for A-to-B transport) or basolateral (for B-to-A transport) compartment in the absence or presence of various concentrations of Compound (I-1) or Ko143. At preset time points, 0.05 mL aliquot of receiving solutions are sampled from the basolateral side (for A-to-B transport) or from the apical side (for B-to-A transport), and replaced immediately with an equal amount of fresh transport media except at the last time point (the end of the incubation). The radioactivity in each sample is measured by 1450 MicroBeta TriLux, a microplate scintillation and luminescence counter (PerkinElmer Life Sciences). Radioactivity (3H) of the dosing solution is measured and used to calculate the initial donor concentration of the substrate.