• Applications of Bile Acid Transporters in Drug Delivery and Enterohepatic Circulation Assessment

      Vivian, Diana; Polli, James E. (2014)
      The objective of this dissertation was to investigate the application of modified bile acids for the assessment of bile acid enterohepatic circulation and for prodrug development. Bile Acid Malabsorption (BAM) results in excess fecal bile acid levels and chronic, intermittent diarrhea. Although BAM may account for up to 30% of patients diagnosed as irritable bowel syndrome (IBS-D), current methods of diagnosis are limited, and BAM is thought to be underdiagnosed. To address this gap, two trifluorinated bile acids were developed for use in fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. CA-lys-TFA was synthesized by conjugating trifluoroacetyl lysine to cholic acid. CA-lys-TFA was a potent inhibitor and substrate of both the apical sodium dependent bile acid transporter and the Na+/taurocholate cotransporting polypeptide. Stability results were favorable in most conditions tested, but in choloylglycine hydrolase (CGH), the compound showed deconjugation. A preliminary pharmacokinetic analysis showed that accumulation of CA-lys-TFA in the gallbladder was maximal within 4 to 7 h. In vivo mouse imaging after oral dosing showed reproducible, clear <super>19</super>F signal from the gallbladder. This represents the first report of in vivo imaging of an orally absorbed drug using <super>19</super>F MRI. To improve upon CA-lys-TFA, CA-sar-TFMA was synthesized as potentially more stable against deconjugation in the intestine. CA-sar-TFMA was a potent inhibitor and substrate of ASBT and NTCP. Contrary to CA-lys-TFA, CA-sar-TFMA showed no degradation when exposed to CGH, and was successfully imaged in the mouse gallbladder. Both CA-lys-TFA and CA-sar-TFMA showed significantly lower concentrations in the gallbladders of Asbt-deficient mice (Slc10A2-/-), which have impaired bile acid transport relative to their WT littermates. These findings suggest that fluorinated non-radioactive bile acid analogues have potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport. Additionally, two bile acid prodrugs of floxuridine were developed to target the liver through NTCP uptake. Finally, a model of substrate translocation in the presence of an inhibitor was derived to determine the impact of microrate constants on K<sub>m</sub>, K<sub>i</sub>, and V<sub>max</sub>.
    • Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake

      Smith, Aaron Dennison; Wilks, Angela (2015)
      Bacterial pathogens require iron for their survival and virulence and have evolved multiple mechanisms to acquire this scarce micro-nutrient. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa acquires heme as an iron source through the Phu (Pseudomonas heme utilization) and Has (Heme assimilation system) systems. The studies herein detail the initial purification and characterization of the outer membrane (OM) HasR and PhuR receptors. A series of site-directed mutagenesis and spectroscopic studies confirmed HasR, in keeping with previously characterized OM receptors, coordinates heme through the conserved N-terminal plug His-221 and His-624 of the surface exposed FRAP-loop. In contrast PhuR coordinates heme through His-124 and Tyr-519 ligands not previously reported in OM receptors but associated with high affinity heme binding proteins. In vivo studies utilizing a combination of bacterial genetics, isotopic labeling (13C-heme), and qRT-PCR further revealed that both receptors are required for optimal heme uptake. However, whereas deletion of hasR leads to an inability to regulate heme uptake, loss of PhuR results in decreased efficiency in heme uptake, despite a significant up regulation in HasR protein levels. The results are consistent with PhuR being the major heme uptake receptor, while HasR senses and regulates extracellular heme uptake. Thus PhuR and HasR represent non-redundant receptors required for accessing and regulating heme uptake across a wide range of physiological conditions found upon infection. The research presented herein also involved optimization of the ABC-transporter ShuUV along with the soluble periplasmic heme binding ShuT proteins from Shigella dysenteriae, which are involved in the transport of heme across the cytoplasmic membrane and into the cell. By generating and screening a series of expression constructs we were able to obtain a construct that resulted in increased expression levels of ShuUV homodimer. Reconstitution of ShuUV in lipososmes with heme loaded ShuT trapped in the interior of the liposome gave a functional system that could transport heme on activation with ATP. Taken together, the current research lays the foundation for future spectroscopic and structural studies aimed at understanding the molecular mechanisms of membrane bound heme transport proteins.
    • Delineating Structural Requirements of hASBT: A Rational Approach to Prodrug Design

      Rais, Rana; Polli, James E. (2010)
      Human apical sodium dependent bile acid transporter (hASBT) plays a critical role in the enterohepatic recirculation of bile acids and is a prodrug target to increase drug absorption. hASBT has been studied to determine the requirements for the binding and transport of bile acid conjugates. Methods to screen and evaluate inhibitors or substrates of this transporter were first optimized and developed. Results suggest the maximum concentration of co-solvents such as DMAC, DMF, DMSO, ethanol, and methanol that could facilitate solubilization of low solubility compounds without detrimentally impacting transporter kinetics. Additionally, extraction methods compatible with LC/MS/MS for substrates were determined. To evaluate the structural requirements of hASBT, 35 monoanionic and dianionic bile acid conjugates were synthesized and evaluated to explore the bile acid chemistry space beyond the C-24 region, and its effect on hASBT binding. Conjugates were subjected to hASBT inhibition and subsequent 3D-QSAR model development. Monoanions were potent inhibitors of hASBT. Dianion potency was due to the presence of specific intramolecular hydrogen bonding, which can be denoted a molecular switch, which allowed for increased conjugate hydrophobicity and hence activity. Validation was performed by synthesizing additional compounds and by NMR methods. Uptake studies were performed on a subset of these compounds, entailing the cellular quantification of compounds using LC-MS. hASBT kinetic parameters Km, Vmax and PP were measured for each conjugate. All mono-anionic conjugates were potent substrates while dianions, cations and zwitterions were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross-validation. The best CSP-SAR model for Km included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for Km/normVmax employed one structural and three physicochemical descriptors, indicating hydrophobicity enhanced efficiency. Lastly, to exemplify hASBT as a prodrug target, three analogues of gabapentin were evaluated as substrates and inhibitors of ASBT. The compounds included two monoanionic and one dianionic conjugate. The potential prodrugs possessed high inhibitory potency along with high affinity for hASBT. The prodrugs were catalytically hydrolyzed to yield drug in Caco-2 homogenates. This work overall helps elucidate structural requirements to successfully design prodrugs to target hASBT.
    • Effect of Cadmium Exposure on the Transport System of Organic Cation Transporters and Multidrug and Toxin Extrusion Proteins (OCTs/MATEs)

      Yang, Hong; Shu, Yan, Ph.D. (2019)
      The universal pollution by cadmium (Cd) in our agricultural land and the prevalence of cigarette smoking make the environmental Cd exposure an unneglectable human health concern. While the mechanism of cadmium accumulation has been extensively studied, no explicit mechanism has been reported regarding the elimination of cadmium from the body. On the other hand, whereas Cd exposure has been correlated with a variety of diseases, little is known pertaining to its effect on drug disposition and response in patients. Thus, we aim to delineate the mechanism of cadmium elimination and detoxification and to gain new insights into its effect on xenobiotic disposition and response. The OCTs/MATEs transport system are pair of transporter proteins highly expressed at the basolateral and apical membrane of hepatocytes and renal proximal tubules respectively. Recently, Cd has been identified as a substrate of OCTs, while we determined that MATEs could reduce the toxicity of Cd by serving as its efflux transporters in vitro. In addition, knockout of Mate1 in mice kidney resulted in higher renal toxicity in both chronic and acute Cd intoxication studies. We found that Cd was an inducer of OCT activity while an inhibitor towards MATEs in cells. Consistently, Cd exposure could lead to accumulation of the substrates of these transporters in mouse liver and kidney. Being focused on human (h) OCT2 and MATE1, our mechanistic studies revealed that hOCT2, as compared to hMATE1, was more active in trafficking between the plasma membrane and the cytoplasmic storage pool. Cd exposure could trigger the formation of a protein complex consisting of AKT2, calmodulin and AS160, which could then selectively facilitate the phosphorylation of AKT2 at T309, and initiate the translocation of hOCT2 to the plasma membrane. Altogether, our findings have identified MATE transporters as new contributors for Cd detoxification, and provided foundation to uncover environmental Cd as a previously unrecognized factor for the broad variation in drug disposition and response.
    • Molecular Insight into the Structure, Function, and Regulation of Bile Acid Transport

      Czuba, Lindsay Christine; Swaan, Peter W.; 0000-0001-9142-5706 (2017)
      The human Apical Sodium-dependent Bile Acid Transporter (SLC10A2), also known as hASBT, plays an integral role in the enterohepatic circulation of bile acid and cholesterol homeostasis. As a member of the solute carrier family of membrane transporters, it uses the established Na+ electrochemical gradient as an energy source to reclaim bile salts from the ileum. hASBT has been identified as a promising target for the management of hypercholesterolemia, cholestatic pruritis, and as a prodrug-targeting approach for improved bioavailability of drugs. Limiting the development of such therapeutics, is an incomplete understanding of hASBT's structure. Extensive biochemical and mutagenesis studies for hASBT support a seven transmembrane model. Yet conflicting structures have emerged with the elucidation of the crystal structures of two putative homologues from Neisseria meningitidis (nmAsbt) and Yersinia frederiksenii (yfAsbt). In the absence of a physiological context, the use of their structure as models of the human transporter is limited. In addition to the discrepancy in hASBT's fold, there is limited information regarding the specific proteoforms that are relevant to the functional expression of hASBT and in its regulation. In this work we provide novel molecular insight into the structure, function, and regulation of human ASBT. We contrasted the biochemical, inhibitory, and evolutionary attributes of nmAsbt, yfAsbt, and hASBT and identified their critical differences. The fundamental differences in ion dependency, substrate specificity, and evolutionary context imply divergent structure-function relationships and negate the use of the bacterial transporters as suitable models for hASBT. Additionally, we characterized the role of tyrosine phosphorylation in regulating the functional expression and stability of hASBT. We identified Src family kinases as critical modulators and provide support for hASBT's regulation by phosphatases. As the clinical relevance of PTMs is growing, so too are the number of FDA-approved therapeutics that target these modifications. In this regard, we have made critical advances and gained valuable insight into hASBT's regulation. Finally, we have optimized the biological sample preparation methods and have significantly increased the purity of hASBT samples. When coupled with mass spectrometry analysis, these methods will identify critical proteoforms of hASBT and facilitate a global understanding of its structure-function relationship.