• 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.
    • Inhibition and Substrate Requirements of human Apical Sodium-dependent Bile Acid Transporter (ASBT) and Its Potential as a Prodrug Target

      Zheng, Xiaowan; Polli, James E. (2010)
      The human apical sodium-dependent bile acid transporter (ASBT; SLC10A2) is an important mechanism for intestinal bile acid reabsorption and plays a critical role in bile acid and cholesterol homeostasis. Its physiological role impacts human health and disease. Furthermore, it is a potential candidate for prodrug targeting due to its high transporter capacity and efficiency. However, the understanding of ASBT's structural determinate of binding and translocation is limited. The work in this dissertation was carried out to study the inhibition and substrate requirement of ASBT, and subsequently to optimize the inhibition assay condition. In particular, work aimed to 1) identify FDA-approved drugs that inhibit ASBT and to derive computational models for ASBT inhibition; 2) evaluate the structural requirements of ASBT by 3D-QSAR analysis using aminopyridine and aminophenol conjugates of chenodeoxycholic acid; 3) synthesis and evaluate in vitro the potential of prolonged release prodrugs via targeting ASBT; 4) identify inhibitor concentrations to efficiently screen and measure inhibition constant Ki values against solute carrier transporters; 5) assess compound cytotoxicity on in vitro apparent transporter inhibition. Many FDA-approved drugs from diverse classes, such as the dihydropyridine calcium channel blockers and HMG CoA-reductase inhibitors were found to be ASBT inhibitors. A 3D-QSAR and a Bayesian model were developed using 38 molecules. 3D-QSAR models also were developed using C-24 conjugates. The models concluded that steric and hydrophobic features strongly influenced conjugate interaction with ASBT, and that the relative location of the pyridine nitrogen and substituent groups also modulated binding. Similar values for Ki and Kt indicated that substrate binding to ASBT was the rate-limiting step. In vitro results showed that the bile acid conjugates are potential prolonged release prodrugs with binding affinity for ASBT. Experimental conditions for Ki screening are suggested to use 10-fold the substrate affinity Kt for potent inhibitors and 100-fold Kt for nonpotent inhibitors; for Ki measurement, the inhibitor concentration range should use 0 to estimated Ki via five different inhibitor concentrations, where a low range of inhibitor concentrations can be used. For some drugs, their cytotoxicites contributed to or were associated with apparent transporter inhibition, where cytotoxicity differed between MDCK and HEK cells; cytotoxicity is suggested for future studies. Overall, the work carried out in this dissertation will aid in advancement in future prodrug design that exploits ASBT and made recommendations for the efficiency and quality of transporter inhibition assays in general.
    • Parametrization for empirical force field calculations and a theoretical study of membrane permeability of pyridine derivatives

      Yin, Daxu; MacKerell, Alexander D., Jr. (1997)
      Parameter sets for alkane, alkene, aliphatic fluorine and pyridine derivatives were developed for empirical force field calculations. Parameters were designed to be compatible with CHARMM22 all hydrogen parameter sets for proteins, nucleic acids and lipids. A novel methodology for optimizing Lennard-Jones parameters was developed by including ab initio interaction energies and geometries between helium or neon and model compounds as goal data. Membrane permeability of pyridine derivatives were studied by developing a quantitative structure-activity relationship (QSAR). Physicochemical parameters used in QSAR were obtained from empirical force field calculations and quantum mechanics calculations.
    • 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).
    • The use of conformational sampling in CHARMM protein force field optimization and ligand-based drug design

      Shim, Jihyun; MacKerell, Alexander D., Jr. (2013)
      Sampling of the conformational space of biomolecules in computer simulations allows researchers to investigate atomistic details of biological phenomena such as protein folding and ligand binding. Conformational sampling based on empirical energy functions depends on the force field and is aided by enhanced simulation methods. This thesis discusses conformational sampling methods and force fields, along with application of conformational sampling to force-field optimization and ligand-based drug design. Extensive conformational sampling was performed for small peptides and drug-like molecules using temperature replica-exchange and Hamiltonian replica-exchange molecular dynamics. Obtained conformational ensembles were then used to improve peptide-backbone and side-chain parameters in the CHARMM protein force fields, thereby yielding more accurate conformational properties. Obtained ensembles were also applied to ligand-based drug design where a novel method based on the conformationally sampled pharmacophore approach was used to identify quantitative structure-activity relationships (SARs) of μ opioid receptor ligands. Based on the SARs, we proposed ligand-binding orientations related to receptor activation. The binding orientations were further investigated using simulations of selected ligands bound to the 3-dimensional -opioid receptor structures. Our studies validate ligand-based SARs and show atomistic details of ligand-receptor interactions and the mechanism of µ opioid receptor activation.