Design of novel synthetic MTS conjugates of bile acids for site-directed sulfhydryl labeling of cysteine residues in bile acid binding and transporting proteins

The purpose of this study was to design bile acid-containing methanethiosulfonate (MTS) agents with appropriate physical attributes to effectively modify the cysteine residues present in the human apical sodium-dependent bile acid transporter. Four physical properties including surface area, molecular volume, C log P, and dipole moment were calculated for each semiempirically optimized structure of MTS compounds. The specificity of the synthesized bile acid–MTS conjugate toward native cysteines and putative bile acid interacting domains of hASBT was supported by the effect of 1 mM cholyl-MTS, cholylglycyl-MTS, and 3-amino-cholyl-MTS on uptake activity, that displayed a significant decrease in TCA affinity (KT = 69.9 ± 4.5, 69.01 ± 6.2, and 63.24 ± 0.26 μM and Jmax = 35.8 ± 0.3, 24.03 ± 1.22, 46.49 ± 5.01 pmol mg protein min−1, respectively). These compounds prove to be effective tools in probing the structural and functional effects of cysteine residues in bile acid binding and transporting proteins.