• 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.
    • Three-dimensional QSAR study to improve intestinal absorption of oral drug candidates via ligand-based drug design: Use and relevance of conformationally sampled pharmacophore approach

      Acharya, Chayan; MacKerell, Alexander D., Jr. (2009)
      In the absence of three-dimensional (3D) structures of potential drug targets, ligand-based drug design is one of the popular approaches for drug discovery and lead optimization. 3D quantitative structure-activity relationships (3D QSAR) and pharmacophore modeling are the most important and widely used tools in ligand-based drug design that can provide crucial insight of the nature of the interactions between drug target and ligand molecule and provide predictive models suitable for lead compound optimization. The work presented in this thesis will discuss the features and potential application of recent advances in ligand-based drug design, along with a detailed description of a novel 3D QSAR method based on the conformationally sample pharmacophore (CSP) approach (denoted CSP-SAR).