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Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.

ABSTRACT: To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes.Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting model). The ability of electrophilic derivatives to interact with hASBT was evaluated by 2-aminoethyl-methanethiosulfonate (MTSEA)-biotin labeling of thiol groups in TM7 cysteine mutants.Unlike native BAs, the electrophilic CDCA derivatives specifically inactivated hASBT, but not hOCTN2, and inhibited hASBT in a time- and concentration-dependent fashion. Preincubation of hASBT Cys-mutants in the exofacial half of TM7 with reactive electrophilic probes blocked transporter biotinylation by MTSEA-biotin, similar to 2-(trimethylammonium)ethyl-methanethiosulfonate (MTSET) blocking. This blocking pattern differed from that produced by native BAs, which exposed exofacial TM7 residues, thereby increasing staining.Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the role of TM7 in binding/translocation of bile acids.

SUBMITTER: Gonzalez PM

PROVIDER: S-EPMC5252617 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.

González Pablo M PM Hussainzada Naissan N Swaan Peter W PW Mackerell Alexander D AD Polli James E JE

Pharmaceutical research 20120222 7

<h4>Purpose</h4>To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes.<h4>Methods</h4>Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting ...[more]

PMID: 22354836

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Project description:BACKGROUND:Cholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated. As one of these mechanisms, a decreased expression of the ileal apical sodium-dependent bile acid transporter gene SLC10A2 in gallstone carriers was described previously. In this study the SLC10A2 gene was investigated to identify novel genetic variants and their association with gallstone formation. METHODOLOGY/PRINCIPAL FINDINGS:Study subjects were selected with the presence or absence of gallstones confirmed by ultrasound and medical history. Genomic DNA was obtained from blood leukocytes. Sequence analysis was performed of all six exonic and flanking regions as well as of 2,400 base pairs of the SLC10A2 promoter in a cohort of gallstone carriers and control subjects from Stuttgart, Germany. Genotype frequencies of newly identified genetic variants (n = 6) and known single nucleotide polymorphisms (n = 24) were established using MALDI-TOF mass spectrometry. Six new genetic variants were found within the SLC10A2 gene. Although none of the variants was linked to gallstone disease in the Stuttgart cohort overall, the minor allele of SNP rs9514089 was more prevalent in male non-obese gallstone carriers (p = 0.06680, OR = 11.00). In a separate population from Aachen, Germany, the occurrence of rs9514089 was two-fold higher in gallstone patients (22%) than in corresponding controls (11%) (p = 0.00995, OR = 2.19). In the pooled Aachen/Stuttgart cohort rs9514089 was highly significantly linked to cholelithiasis (p = 0.00767, OR = 2.04). A more frequent occurrence was observed for male gallstone carriers (22%) compared to controls (9%) (p = 0.01017, OR = 2.99), for the total normal weight group (p = 0.00754, OR = 2.90), and for male non-obese gallstone patients (p = 0.01410, OR = 6.85). Moreover, for the minor allele of rs9514089 an association with low plasma cholesterol levels was found especially in gallstone carriers (p = 0.05). CONCLUSIONS/SIGNIFICANCE:We have identified SLC10A2 as a novel susceptibility gene for cholelithiasis in humans. Comprehensive statistical analysis provides strong evidence that rs9514089 is a genetic determinant especially in male non-obese gallstone carriers. The minor allele of rs9514089 is related to differences in plasma cholesterol levels among the subjects.

Project description:Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3?-hydroxyl (-OH) group on the cholestane ring to the common 3?-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3?-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3?-OH group on BAs binding and translocation by hASBT. Ten 3?-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3?-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT. Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3?-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH "on-" or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3?-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3?-OH group. Results have implications for BA prodrug design.

Dataset Information

Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.

Publications

Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.

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