ABCMdb

PMID: 22583617

Misawa T, Hayashi H, Makishima M, Sugiyama Y, Hashimoto Y
E297G mutated bile salt export pump (BSEP) function enhancers derived from GW4064: structural development study and separation from farnesoid X receptor-agonistic activity.
Bioorg Med Chem Lett. 2012 Jun 15;22(12):3962-6. doi: 10.1016/j.bmcl.2012.04.099. Epub 2012 Apr 30., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Several BSEP mutants, including Glu297Gly (E297G) and Asp482Gly (D482G), cause progressive familial intrahepatic cholestasis type 2. Login to comment 2 ABCB11 p.Glu297Gly We previously found that compounds based on GW4064, a representative farnesoid X receptor (FXR) agonist, enhanced E297G BSEP transport activity. Login to comment 3 ABCB11 p.Glu297Gly Here, we conducted a structure-activity relationship analysis of GW4064 derivatives aimed at separating E297G BSEP-function-promoting activity and FXR-agonistic activity. Among newly synthesized reversed-amide derivatives of previously reported GW4064 analogs 2a-2f, we identified 7c as a selective BSEP function enhancer. Login to comment 5 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Bile salt export pump (BSEP) is a member of the ATP-binding cassette transmembrane transporter family and mediates the biliary excretion of monovalent bile acids (such as taurocholate) from hepatocytes.1-3 It is well known that several BSEP mutants cause progressive familial intrahepatic cholestasis type 2 (PFIC2) which is a recessive hereditary liver disease characterized by cholestasis and jaundice in the first year of life, leading to cirrhosis and death before adulthood unless liver transplantation is carried out.4 Among the PFIC2-causing mutations, two missense mutations, that is, E297G (Glu297Gly) and D482G (Asp482Gly), are frequently observed in PFIC2 patients.5,6 The E297G mutant is considered to be folding-defective (misfolded), and as a result, this mutant BSEP is retained within the cell on endoplasmic reticulum (ER), and does not acquire the usual golgi-related glycosylation. Login to comment 6 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly 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 are correctly localized.7 Furthermore, Hayashi et al. reported that sodium 4-phenylbutyric 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.6-8 In our previous work, we found that bile acids showed moderate E297G BSEP-function-promoting activity, and chenodeoxycholic acid (CDCA) was the most potent function-promoter among the tested bile acids.9 Furthermore, compounds based on GW4064 [a representative farnesoid X receptor (FXR) agonist10 ] ( Fig. 1) enhanced the BSEP transport activity. Among the tested compounds, compound 2d possessed the most potent E297G BSEP-function-promoting activity at 10 lM (Table 2). Login to comment 9 ABCB11 p.Glu297Gly FXR is a well-characterized member of the so-called metabolic subfamily of nuclear receptors, and is a transcriptional sensor for bile acids.12,13 It is well known that FXR-agonists such as CDCA and GW4064 increase endogenous BSEP mRNA level and lower the bile acid pool.14 We considered that the contribution of FXR-agonistic activity to the improvement of BSEP function in our assay system was negligible, because we had calculated the ratio of the amount of [3 H]taurocholate ([3 H]TC) accumulated in MDCK II cells expressing WT or E297G BSEP versus that in the MDCK II cells expressing GFP to confirm the effect of GW4064 derivatives on the function of BSEP. Login to comment 18 ABCB11 p.Glu297Gly Structure of GW4064 and E297G BSEP function enhancers. Login to comment 19 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Table 1 E297G BSEP-function-promoting activity and FXR-agonistic activity of bile acids Compd R1 R2 Accumulation of [3 H]TCa (%) at 100 lM FXR-agonistic activity EC50 c (lM) CA (3) OH OH 40b NAd DCA (4) H OH 65b 50 CDCA (5) b-OH H 46b 27 UDCA (6) a-OH H 43b NAd a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment 22 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Table 2 FXR-agonistic activity of E297G BSEP function enhancers Compd R1 R2 Accumulation of [3 H]TCa (%) at 10 lM FXR-agonistic activity EC50 c (lM) and efficacyd (%) CDCA (5) 50b 27 (41) GW4064 (1) 33b 0.07 (100) 2a 3-COOH H 111b 3.0 (61) 2b 3-COOH Me 66b 3.1 (45) 2c 3-COOH n-Bu 49b 1.1 (43) 2d 3-COOH Bn 31b 2.5 (31) 2e 3-COOH Phenethyl 53b 0.84 (30) 2f 3-COOH 1-Naphthyl 59b 3.0 (28) 2g 2-COOH Bn 80b NAe 2h 4-COOH Bn 35b 2.6 (41) 2i 3-COOMe Bn 93b NAe a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment 29 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly One was an accumulation assay to evaluate the E297G BSEP-function-promoting activity, using Madin-Darby canine kidney II (MDCKII) cells expressing Na+ -taurocholate cotransporting polypeptide (NTCP) and E297G BSEP, as described previously.9 With this method, we evaluated the amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP; this accumulation is determined by several parameters, including the cellular efflux of [3 H]TC via BSEP. Login to comment 31 ABCB11 p.Glu297Gly We previously reported that these bile acids possess moderate E297G BSEP-function-promoting activity.9 Compounds 4 and 5 showed weak FXR-agonistic activity, whereas compounds 3 and 6 showed no FXR-agonistic activity (Table 1). Login to comment 32 ABCB11 p.Glu297Gly These results suggest that E297G BSEP-function-promoting activity can be separated from FXR-agonistic activity. Login to comment 38 ABCB11 p.Glu297Gly Next, we investigated the FXR-agonistic activity of our previously reported E297G BSEP function enhancers derived from GW4064 (2a-2i, Table 2). Login to comment 39 ABCB11 p.Glu297Gly E297G BSEP function enhancers 2a-f and 2h showed weak FXR-agonistic activity, while 2g and 2i were inactive. Login to comment 47 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly In the case of GW4064, a brief SAR report from GSK researchers disclosed that a carboxyl group is essential and is preferably located at the 3-position (meta-position) for Table 3 E297G BSEP-function-promoting activity and FXR-agonistic activity of 7a-7e Compd R Accumulation of [3 H]TCa (%) at 10 lM FXR-agonistic activity EC50 (lM) and efficacyb (%) CDCA 50 27 (41) GW4064 33 0.07 (100) 2d 31 2.5 (31) 7a H 75 NAc 7b Me 60 NAc 7c n-Bu 42 NAc 7d Bn 50 >10 7e 1-Naphthyl 59 0.41 (35) a The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment 51 ABCB11 p.Glu297Gly Concentration dependency of the effect of 2d, 7a, and 7c-7d on [3 H]TC accumulation in MDCK II cells expressing E297G BSEP. Login to comment 52 ABCB11 p.Glu297Gly The amount of [3 H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%. Login to comment 54 ABCB11 p.Glu297Gly ortho-Isomer 2g and ester derivative 2i showed neither E297G BSEP-function-promoting nor FXR-agonistic activity, whereas the para-isomer (2h) showed these activities with similar potency to the meta-isomer 2d. Login to comment 56 ABCB11 p.Glu297Gly As expected, E297G BSEP function enhancers (2a-2f, 2h) also showed FXR-agonistic activity (Table 2). Login to comment 57 ABCB11 p.Glu297Gly As shown in Table 2, substitution of the double bond at the central portion of GW4064 (1) to an amide bond, that is, compound 2d, had no effect on E297G BSEP-function-promoting activity, but decreased the potency of FXR-agonistic activity, that is, the EC50 values for GW4064 (1) and 2d are 0.07 lM and 2.5 lM, respectively. Login to comment 62 ABCB11 p.Glu297Gly So, we speculated that the N-substituted amide-type central portion of the molecule might be the key to separating E297G BSEP-function-promoting activity from FXR-agonistic activity. Login to comment 68 ABCB11 p.Glu297Gly We evaluated the E297G BSEP-function-promoting activity and FXR-agonistic activity of the prepared reversed-amide derivatives 7a-7e (Table 3 and Fig. 3). Login to comment 69 ABCB11 p.Glu297Gly These compounds possessed moderate to potent E297G BSEP-function-promoting activity. Among the reversed-amide derivatives, N-butyl derivative 7c possessed the most potent activity (accumulation of [3 H]TC was reduced to 42% and 59% at 10 lM and 1 lM, respectively, Table 3 and Fig. 3). Login to comment 71 ABCB11 p.Glu297Gly In summary, we confirmed that bile acids and 2a-2f exhibit both FXR-agonistic activity and E297G BSEP-function-promoting activity. Login to comment