ABCB1 p.Phe336Ala
| Predicted by SNAP2: | A: D (63%), C: D (59%), D: D (85%), E: D (80%), G: D (75%), H: D (80%), I: D (66%), K: D (85%), L: N (57%), M: N (61%), N: D (80%), P: D (85%), Q: D (75%), R: D (85%), S: D (66%), T: D (71%), V: D (71%), W: D (71%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, 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
11 For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillari- dinA, 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.
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ABCB1 p.Phe336Ala 23104431:11:238
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.Phe336Ala 23104431:62:55
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.Phe336Ala 23104431:122:109
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.Phe336Ala 23104431:132:40
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.Phe336Ala 23104431:135:38
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.Phe336Ala 23104431:180:98
status: NEW192 Wild-type, F336A (A), and I340A (B) mutant P-gp membrane vesicles were incubated with 100nM NMQ (containing 10nM [3 H]-NMQ) in the absence or presence of indicated concentrations of cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol.
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ABCB1 p.Phe336Ala 23104431:192:11
status: NEW194 The data points have been shown by (ߦ) for wild-type, (a0;) for F336A mutant, and (b2;) for I340A mutant P-gp.
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ABCB1 p.Phe336Ala 23104431:194:74
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.Phe336Ala 23104431:202:58
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.Phe336Ala 23104431:208:628
status: NEW214 More remarkable were the inhibitory affinities of F336A and I340A, which were significantly different in five cases.
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ABCB1 p.Phe336Ala 23104431:214:50
status: NEW[hide] Convallatoxin: a new P-glycoprotein substrate. Eur J Pharmacol. 2014 Dec 5;744:18-27. doi: 10.1016/j.ejphar.2014.09.031. Epub 2014 Sep 28. Gozalpour E, Greupink R, Bilos A, Verweij V, van den Heuvel JJ, Masereeuw R, Russel FG, Koenderink JB
Convallatoxin: a new P-glycoprotein substrate.
Eur J Pharmacol. 2014 Dec 5;744:18-27. doi: 10.1016/j.ejphar.2014.09.031. Epub 2014 Sep 28., [PMID:25264938]
Abstract [show]
Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1+/-0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp.
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No. Sentence Comment
56 Nine different P-gp mutants, I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A, were produced and sequencing of full-length P-gp cDNA was used to confirm all mutations (Gozalpour et al., 2013).
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ABCB1 p.Phe336Ala 25264938:56:36
status: NEW154 Nine amino acids were replaced by alanine (I306A, F336A, I340A, F343A, F728A, F942A, T945A, L975A, and V982A) and P-gp mutants were expressed in HEK293 cells to produce membrane vesicles.
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ABCB1 p.Phe336Ala 25264938:154:50
status: NEW168 The transport activity of F336A, F942A, T945A and L975A for NMQ ranged from 49% to 57%, whereas I340A showed increased activity of 120% and V982A had about the same activity as wild type (Fig. 5B).
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ABCB1 p.Phe336Ala 25264938:168:26
status: NEW170 Convallatoxin and NMQ transport activity were not significantly different for I306A, F336A, I340A, F728A, F942A, T945A, and L975A (Fig. 5C), whereas they differed significantly for F343A and V982A (Fig. 5D and E).
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ABCB1 p.Phe336Ala 25264938:170:85
status: NEW254 The transport activity of F336A, F942A, T945A, L975A and V982A, were conserved (45-100% of wild type) (Fig. 5B) similar to our previous results (Gozalpour et al., 2013).
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ABCB1 p.Phe336Ala 25264938:254:26
status: NEW255 Convallatoxin transport activity of most mutants (I306A, F336A, I340A, F728A, F942A, T945, and L975) was similar to that of NMQ.
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ABCB1 p.Phe336Ala 25264938:255:57
status: NEW