ABCMdb

PMID: 22464344

Misawa T, Hayashi H, Sugiyama Y, Hashimoto Y
Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2.
Bioorg Med Chem. 2012 May 1;20(9):2940-9. doi: 10.1016/j.bmc.2012.03.016. Epub 2012 Mar 14., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCB11 p.Glu297Gly Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2 Takashi Misawa a , Hisamitsu Hayashi b , Yuichi Sugiyama b,d1; , Yuichi Hashimoto a,d1; a Institute of Molecular and Cellular Biosciencies, The University of Tokyo, 1-1-1 Bunkyo-ku, Tokyo 113-0032, Japan b Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan a r t i c l e i n f o Article history: Received 13 February 2012 Revised 5 March 2012 Accepted 5 March 2012 Available online 14 March 2012 Keywords: Bile salt export pump Bile acid Farnesoid X receptor Mutant a b s t r a c t Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by hereditary mutations of bile salt export pump (BSEP), such as E297G BSEP, which is a folding-defective mutant that is unable to traffic beyond the endoplasmic reticulum (ER). Login to comment 1 ABCB11 p.Glu297Gly 4-Phenylbutyric acid (4-PBA) enhances the cell surface expression and transport capacity of E297G BSEP, but has a relatively high dose (1 mM or more) is required to show the effect. Login to comment 2 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Here, we show that bile acids possibly act as pharmacological chaperones, promoting the proper folding and trafficking of E297G BSEP. We also describe the discovery and structural development of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP. Login to comment 8 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Genomic analysis of PFIC2 patients has revealed many kinds of missense, premature termination, frame-shift and splicing-junction mutations in the BSEP gene.3 Among them, two missense mutations, E297G (Glu297Gly) and D482G (Asp482Gly), are frequently observed in PFIC2 patients.4,5 Generally, wild-type (WT) BSEP is synthesized and folded at the endoplasmic reticulum (ER). Login to comment 10 ABCB11 p.Glu297Gly But, the E297G mutant is considered to be folding-defective (misfolded). Login to comment 12 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Hayashi et al. reported that the E297G and D482G mutations cause a reduction of BSEP on plasma membrane, but importantly, they found that the transport function of both mutated BSEPs is almost normal if they localized correctly.5 Thus, the folding-defective nature, linked to abnormal trafficking (retention at ER), of E297G mutant lies at the core of the etiology of PFIC2, at least in part. Login to comment 13 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly If E297G BSEP could be induced to fold properly, the mutant protein could be transported to the correct localization, where it should be functional, providing a promising strategy for treatment of E297G BSEP-related PFIC2. Login to comment 14 ABCB11 p.Glu297Gly Hayashi et al. reported that sodium 4-phenylbutylic acid (4-PBA) enhanced the cell-surface expression and transport capacity of E297G BSEP by prolonging the half-life of cell surface-resident BSEP, through modulating its ubiquitination status and AP2-mediated internalization.5-7 Although 4-PBA can be beneficial for the treatment for PFIC2, a relatively high dose (1 mM or more) is required to rescue the function of the mutant BSEP in vivo. Login to comment 16 ABCB11 p.Glu297Gly Based on this background, we searched for novel compounds able to enhance the transport capacity of E297G BSEP at lower doses. Login to comment 21 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Bioorganic & Medicinal Chemistry 20 (2012) 2940-2949 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc target protein and induces or promotes proper folding and trafficking of the protein.13 The advantages of pharmacological chaperones are considered to include high specificity and efficacy at much lower dose levels than 4-PBA.13 In the cases of some folding-defective mutant proteins, including mutated rhodopsin, it has been shown that their ligands (substrates, inhibitors, modulators, etc.) act as pharmacological chaperones.8 Therefore, we hoped that bile acids, which are substrates of BSEP, might act as pharmacological chaperones of E297G BSEP, consequently enhancing the cell-surface expression and transport capacity of E297G BSEP. Login to comment 22 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Here, we show that bile acids do act as pharmacological chaperones of E297G BSEP. We also describe the discovery and structural development of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP. Login to comment 25 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Effects of bile acids on [3 H]TC accumulation To screen compounds that enhance the transport capacity of E297G BSEP, we established a functional assay system, using Madin-Darby canine kidney II (MDCKII) cells expressing Na+ -taurocholate cotransporting polypeptide (NTCP) and WT or E297G BSEP. Login to comment 26 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The amount of [3 H]taurocholate ([3 H]TC) accumulation in cells expressing E297G BSEP was higher than that in cells 0.00 0.50 1.00 1.50 2.00 2.50 WT E297G CA 0uM 10uM 100uM [ 3 H]TC accumulation (BSEP cells/GFP cells) 0&#b5;M 10&#b5;M 100&#b5;M Figure 1. Concentration-dependence of the effect of cholic acid (CA) on [3 H]TC accumulation in MDCK II cells expressing wild type (WT) BSEP or E297G BSEP. Vertical scale: The amounts of [3 H]TC accumulated in the MDCK II cells transfected with WT BSEP, E297G BSEP or GFP were measured by means of radioactivity (dpm). Login to comment 28 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Table 1 Decreasing effect of bile acids on [3 H]TC accumulation O O R2 R3 H R3 H H H HO R1 H H H HO R1 Compd R1 R2 R3 Accumulation of [3 H] TC (%) 100 lM 10 lM Cholic acid (CA) OH OH OH 40 73 Glycocholic acid (GC) OH OH Glycine 108 Taurocholic acid (TC) OH OH Taurine 105 Deoxycholic acid (DCA) H OH OH 65 Chenodeoxycholic acid (CDCA) b-OH H OH 46 50 Ursodeoxycholic acid (UDCA) a-OH H OH 43 76 Table 2 Decreasing effect of GW4064 and derivatives on [3 H]TC accumulation N O O N R1 R4 R1 R1 N R3 R2 O R3 Compd R1 R2 R3 R4 Accumulation of [3 H]TC (%) at 10 lM CDCA 50 GW4064 33 6g Cl Cl 3-COOH H 111 6a Cl Cl 3-COOH Me 66 6b Cl Cl 3-COOH n-Bu 49 6c Cl Cl 3-COOH Bn 31 6d Cl Cl 3-COOH Phenethyl 53 6e Cl Cl 3-COOH 1-Naphthyl 59 6f Cl Cl 3-COOH 2-Naphthyl 70 15a H Cl 3-COOH Bn 64 15b Cl H 3-COOH Bn 85 15c Me Cl 3-COOH Bn 93 15d Cl Cl 2-COOH Bn 80 15e Cl Cl 4-COOH Bn 35 5c Cl Cl 3-COOMe Bn 93 expressing WT BSEP because of the cellular efflux function of [3 H]TC by BSEP and the lower cell-surface expression of E297G BSEP compared with WT BSEP (Fig. 1).14 We first examined the effect of several BSEP substrates and other related bile acids, that is, cholic acid (CA), glycocholic acid (GC), taurocholic acid (TC), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA), on the function of E297G BSEP. Login to comment 31 ABCB11 p.Glu297Gly 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 3 4 0 1 3 10 Concentration (&#b5;M) 6c [ 3 H]TC accumulation (BSEP cells/GFP cells) 0 0.5 1 1.5 2 2.5 3 3.5 1 2 3 4 0 1 3 10 Concentration (&#b5;M) GW4064 [ 3 H]TC accumulation (BSEP cells/GFP cells) (a) (b) Figure 3. Concentration-dependence of the effects of GW4064 (a), and 6c (b) on [3 H]TC accumulation in MDCK II cells expressing E297G BSEP. Vertical scale: same as the legend of Figure 1. Login to comment 39 ABCB11 p.Glu297Gly Effect of 4-PBA and 6c on [3 H]TC transport across the apical membrane in monolayers of MDCK II cells expressing E297G BSEP. Login to comment 43 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly We speculated that bile acids increased the cell-surface expression of E297G BSEP by acting as pharmacological chaperones, thereby decreasing [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment 49 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly CDCA is an endogenous FXR ligand,15,16 and acts as a signaling molecule that governs lipid, steroid, and cholesterol homeostasis.17 FXR is involved in possesses such as glucose utilization, inflammation, and carcinogenesis.18,19 After deorphanization of FXR in 1999, extensive studies were directed to the creation of FXRligands thatcouldmodulateFXR-mediatedspecificgene expression, and several steroidal or non-steroidal FXR ligands have been reported (Fig. 2).20-22 We hypothesized that non-steroidal FXR ligands might act as a structural equivalent of CDCA and enhance the transport capacity of E297G BSEP. First, we investigated whether FXR ligands enhance the transport capacity of E297G BSEP. We found that GW4064, which is a potent non-steroidal agonist for FXR, dose-dependently decreased [3 H]TC accumulation (Fig. 3a). Login to comment 50 ABCB11 p.Glu297Gly GW4064has theadvantagethatitsbasic architecture isdistinctfrom that of CDCA, and it shows no activity towards other nuclear receptors, including the retinoic acid receptor.20 GW4064 seemed to be superior to CDCA in its efficacy to decrease [3 H]TC accumulation in cells expressing E297G BSEP (Table 2), and was selected as a lead compound for further structural development. Login to comment 52 ABCB11 p.Glu297Gly Structure-activity relationship of GW4064 derivatives Kainuma and co-workers reported that the double bond of GW4064 can be replaced with a carbamoyl moiety without loss of FXR activity.23 Similarly, we designed and prepared secondary amide 6g, N-methyl amide 6a, N-butyl amide 6b, N-benzyl amide 6c, N-phenethyl amide 6d, and N-naphthyl amide 6e and 6f derivatives, and examined whether these derivatives decrease [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment 66 ABCB11 p.Glu297Gly As shown in Table 2, ester derivative 5c had no effect on [3 H]TC accumulation in cells expressing E297G BSEP at 10 lM. Login to comment 68 ABCB11 p.Glu297Gly These results indicate that a carboxyl substituent at the meta- or para-position is important for interaction with E297G BSEP. Login to comment 72 ABCB11 p.Glu297Gly Transcellular transport assay Finally, we evaluated the effectiveness of 6c in a transcellular transport assay with MDCK II cells expressing E297G BSEP. Login to comment 77 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Conclusion We focused on the idea that the transport capacity of E297G BSEP might be increased by pharmacological chaperones that promote proper trafficking of the mutant BSEP. First, we established that bile acids decrease [3 H]TC accumulation in cells expressing E297G BSEP. Login to comment 80 ABCB11 p.Glu297Gly Further studies to examine in detail the molecular mechanism(s) of the enhancement of E297G BSEP trafficking and the effects of these compounds on other BSEP mutants are in progress. Login to comment 91 ABCB11 p.Glu297Gly The BD Adeno-X-Adenoviral Expression System (BD Biosciences, Palo Alto, CA) was used to establish human WT and E297G BSEP recombinant adenoviruses as previously described.14 As a control, recombinant adenovirus containing green fluorescence protein (GFP) was used. Login to comment 93 ABCB11 p.Glu297Gly Generation of recombinant adenovirus The recombinant adenoviruses containing wild-type BSEP, E297G BSEP, or GFP were constructed as described previously.14 The BSEP cDNA was amplified via polymerase chain reaction(PCR) with Ex-Taq (Takara Bio, Shiga, Japan) from the commercially available cDNA library (Clontech, PaloAlto, CA). Login to comment 103 ABCB11 p.Glu297Gly After 2 days` culture, confluent cells were infected with recombinant adenovirus containing cDNA for WT, E297G, BSEP or GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with compounds. Login to comment 107 ABCB11 p.Glu297Gly After 1 day of culture, confluent cells were infected with recombinant adenovirus containing cDNA for WT, E297G BSEP or GFP at 50 MOI. Cells were cultured for 24 h after infection and subsequently treated with compounds. Login to comment