Browsing by Author "Polli, James E."

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    Impact of impurity on kinetic estimates from transport and inhibition studies
    (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2008) Gonzalez, Pablo; Polli, James E.
    Although in vitro transport/inhibition studies are commonly performed on impure drug candidates to screen for pharmacokinetic properties in early development, quantitative guidelines concerning acceptable impurity levels are lacking. The broad goal was to derive models for the effect of impurity on transport and inhibition studies and identify the maximum allowable impurity level that does not bias assay results. Models were derived, and simulations were performed to assess the impact of impurity on substrate properties K-t and J(max) and inhibition K-i. Simulation results were experimentally challenged with a known amount of impurity, using the intestinal bile acid transporter as a model system. For substrate uptake studies, glycocholate served as substrate and was contaminated with either a very strong, strong, or moderate impurity (i.e., taurolithocholate, chenodeoxycholate, or ursodeoxycholate, respectively). For inhibition studies, taurocholate and glycocholate together was the substrate/inhibitor pair, where glycocholate was contaminated with taurolithocholate. There was high agreement between simulation results and experimental observations. It is not surprising that, in the inhibition assay, potent impurity caused test compound to appear more potent than the true potency of the test compound (i.e., reduced inhibitory K-i). However, results in the transport scenario surprisingly indicated that potent impurity did not diminish test compound potency but, rather, increased substrate potency (i.e., reduced Michaelis-Menten substrate K-t). In general, less than 2.5% impurity is a reasonable target, provided the impurity is less than 10-fold more potent than test compound. Study results indicate that careful consideration of possible impurity effect is needed when quantitative structure-activity relationship analysis cannot explain high compound potency from transport or inhibition studies.
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    Inhibition Requirements of the Human Apical Sodium-Dependent Bile Acid Transporter (hASBT) Using Aminopiperidine Conjugates of glutamyl-Bile Acids
    (2009) Gonzalez, Pablo M.; Acharya, Chayan; MacKerell, Alexander D., Jr.; Polli, James E.
    Synthesize aminopiperidine conjugates of glutamyl-bile acids (glu-BAs) and develop a hASBT inhibition model using the conformationally sampled pharmacophore (CSP) approach.
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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
    (2012) Gonzalez, Pablo M.; Hussainzada, Naissan; Swaan, Peter W.; MacKerell, Alexander D., Jr.; Polli, James E.
    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.
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    Structural Requirements of the Human Sodium-Dependent Bile Acid Transporter (hASBT): Role of 3-and 7-OH Moieties on Binding and Translocation of Bile Acids
    (2014) Gonzalez, Pablo M.; Lagos, Carlos F.; Ward, Weslyn C.; Polli, James E.
    Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3 beta-hydroxyl (-OH) group on the cholestane ring to the common 3 alpha-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 beta-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3 alpha-OH group on BAs binding and translocation by hASBT. Ten 3 beta-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 alpha-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 alpha-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 alpha-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 alpha-OH group. Results have implications for BA prodrug design.