ABCB1 p.Leu65Ala
| Predicted by SNAP2: | A: N (72%), C: N (66%), D: N (53%), E: N (61%), F: N (93%), G: N (53%), H: N (61%), I: N (87%), K: N (57%), M: N (93%), N: N (72%), P: N (57%), Q: N (93%), R: N (66%), S: N (78%), T: N (87%), V: N (87%), W: N (82%), Y: N (78%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] Interaction of digitalis-like compounds with p-gly... Toxicol Sci. 2013 Feb;131(2):502-11. doi: 10.1093/toxsci/kfs307. Epub 2012 Oct 26. Gozalpour E, Wittgen HG, van den Heuvel JJ, Greupink R, Russel FG, Koenderink JB
Interaction of digitalis-like compounds with p-glycoprotein.
Toxicol Sci. 2013 Feb;131(2):502-11. doi: 10.1093/toxsci/kfs307. Epub 2012 Oct 26., [PMID:23104431]
Abstract [show]
Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12beta seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a delta-lactone ring and a sugar moiety at 3beta of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5beta and 19, whereas inhibition is decreased by those at positions 1beta, 11alpha, 12beta, and 16beta. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.
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No. Sentence Comment
46 However, transport activity was preserved in L65A, I306A, I340A, F942A, and V982A.
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ABCB1 p.Leu65Ala 23104431:46:45
status: NEW62 Ten different P-gp mutants were produced: L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A and all mutations were confirmed by sequencing of full-length P-gp cDNA.
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ABCB1 p.Leu65Ala 23104431:62:42
status: NEW122 All the indicated amino acids were replaced by alanine to remove the side chain of the residue (L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A).
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ABCB1 p.Leu65Ala 23104431:122:96
status: NEW132 NMQ transport activity of mutants L65A, F336A, I340A, F942A, andV982A as compared with wild-type P-gp ranged from 60 to 150%, whereas NMQ transport activity of I306A, F343A, F728A, T945A, and L975A varied between 8 and 30%.
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ABCB1 p.Leu65Ala 23104431:132:34
status: NEW135 In addition, five P-gp mutants (L65A, F336A, I340A, F942A, and V982A), for which NMQ transport activity was at least 50% of wild-type transport, were selected.
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ABCB1 p.Leu65Ala 23104431:135:32
status: NEW138 Comparing the IC50 value of the mutants with those of the wild type (mutant IC50 /wild-type IC50 ), L65A, and V982A showed similar values as wild type (0.6-2.2).
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ABCB1 p.Leu65Ala 23104431:138:100
status: NEW180 Fig. 5.ߓ Western blot analysis (A) and NMQ transport activity of wild type and L65A, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A mutant P-gp (B).
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ABCB1 p.Leu65Ala 23104431:180:85
status: NEW202 NMQ transport activity of the second group mutants (L65A, F336A, I340A, F942A, and V982A) was not significantly different from that of the wild-type P-gp (60-150%).
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ABCB1 p.Leu65Ala 23104431:202:52
status: NEW208 Table 1 The IC50 Values of DLCs Against Wild-Type and Mutant P-gp-Mediated [3 H]-NMQ Transport P-gp Cymarin Digitoxin Digoxin Peruvoside Proscillaridin A Strophanthidol IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 IC50 (&#b5;M) RIC50 Wild type 432ߙ&#b1;ߙ90 9ߙ&#b1;ߙ2.1 188ߙ&#b1;ߙ24 214ߙ&#b1;ߙ41 25ߙ&#b1;ߙ3.5 242ߙ&#b1;ߙ61 L65A 800ߙ&#b1;ߙ99 1.9 17ߙ&#b1;ߙ3.4 1.9 217ߙ&#b1;ߙ33 1.2 469ߙ&#b1;ߙ73* 2.2 48ߙ&#b1;ߙ7.2 1.9 186ߙ&#b1;ߙ18 0.8 F336A 979ߙ&#b1;ߙ48 2.3 14ߙ&#b1;ߙ1.9 1.6 524ߙ&#b1;ߙ114 2.8 528ߙ&#b1;ߙ92** 2.5 111ߙ&#b1;ߙ19** 4.4 291ߙ&#b1;ߙ45 1.2 I340A 1181ߙ&#b1;ߙ103** 2.7 19ߙ&#b1;ߙ4.3 2.0 439ߙ&#b1;ߙ138 2.3 527ߙ&#b1;ߙ37** 2.5 79ߙ&#b1;ߙ21* 3.1 156ߙ&#b1;ߙ14 0.6 F942A 821ߙ&#b1;ߙ256 1.9 8ߙ&#b1;ߙ1.0 0.9 558ߙ&#b1;ߙ145 3.0 273ߙ&#b1;ߙ29 1.3 18ߙ&#b1;ߙ1 0.7 187ߙ&#b1;ߙ24 0.8 V982A 620ߙ&#b1;ߙ106 1.4 12ߙ&#b1;ߙ2.1 1.3 345ߙ&#b1;ߙ73 1.8 291ߙ&#b1;ߙ10 1.4 22ߙ&#b1;ߙ3.4 0.9 144ߙ&#b1;ߙ9 0.6 Note.
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ABCB1 p.Leu65Ala 23104431:208:448
status: NEW213 Inhibition of NMQ transport by L65A, F942A, and V982A with six DLCs showed that in only one case the mutation caused a significant difference in the IC50 value (ratio of 2.2) of the DLCs compared with wild-type P-gp.
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ABCB1 p.Leu65Ala 23104431:213:31
status: NEW