• Identification of Drugs of Abuse as Modulators of Drug-Metabolizing Enzymes through Nuclear Receptor-Mediated Mechanisms

      Baaqee, Antonia Tolson; Eddington, Natalie D. (2012)
      To date, the majority of reports discussing opioid-drug interactions focus intensively on characterizing how other drugs affect the metabolic and pharmacokinetic (MPK) profile of opioids, however little has been published regarding the potential for opioids to modulate MPK-based drug-drug interactions (DDIs) involving other commonly co-administered or co-abused drugs. Moreover, virtually no mechanistic evidence has been explored. Thus, the objective of this work was to elucidate how opioids affect the MPK of other drugs, thereby undertaking research from a perspective that has been historically overlooked. Accordingly, the specific aims of this study were to: 1) Screen several different drugs of abuse for nuclear receptor (NR) activation potential, 2) Determine the expression profiles of key drug-metabolizing enzymes (DMEs) or drug transporters for selected drugs in human primary hepatocytes (HPHs), and 3) Characterize the mechanistic roles played by xenoreceptors Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) underlying observed DME modulation. Results: Here we show that several opioids were identified as potential NR activators, and selected drugs of abuse exhibited differential induction profiles at the mRNA level for target genes CYP2B6 and CYP3A4. Overall, for opioid therapies MD and BUP: 1) MD induced the hepatic expression of multiple key DMEs by activating PXR- and CAR-mediated pathways; 2) More specifically, MD treatment resulted in significant nuclear accumulation of adenovirus/enhanced yellow fluorescent protein tagged-hCAR in HPHs, which has been regarded as the initial step of CAR activation, and additional analysis of the two enantiomers of racemic MD, R-(-)-MD (active) and S- (+)-MD (inactive), indicated a lack of stereoselectivity pertaining to MD-mediated DME induction; 3) For BUP, although hPXR-mediated CYP2B6 and CYP3A4 reporter activities were significantly increased in HepG2 cells, treatment with identical concentrations of buprenorphine in HPHs resulted in literally no induction of target gene expression. Taken together, these results provide much-needed mechanistic evidence which demonstrates that MD may be more likely than BUP to modulate CAR- and PXR- mediated DME perturbation during opioid-drug interactions. This research is of great importance to the overall public health industry, particularly to those clinicians and research scientists whom administer MD or BUP as part of opioid maintenance pharmacotherapy.
    • Molecular Mechanisms Underlying the Regulation of Mu Opioid Receptor Function by Protein Kinase C and Histidine Triad Nucleotide Binding Protein 1

      Feng, Bo; Wang, Jia Bei (2010)
      Mu opioid receptor (MOPr) belongs to G protein-coupled receptor superfamily and is the primary target through which opioid drugs exert their biological activities. Multiple lines of evidence support that agonist-induced adaptive changes of MOPr are important molecular mechanisms underlying the development of opioid tolerance and dependence. It has also been shown that phosphorylation of MOPr, especially phosphorylation of C-terminus, plays a critical role in the regulation of receptor adaptive changes. In addition to G protein-coupled receptor kinase (GRK), second messenger protein kinases, including protein kinase C (PKC), are also involved in the phosphorylation of MOPr. Although previous research has shown that PKC is capable of regulating MOPr adaptive changes and the development of opioid tolerance and dependence, functional impacts of PKC-mediated receptor phosphorylation on MOPr signaling remain unclear. To test the hypothesis that PKC-mediated regulation of MOPr signaling is achieved through phosphorylation of the receptor C-terminus. In vitro phosphorylation results demonstrated that PKC is capable of phosphorylating MOPr C-terminus and Ser363 is the primary PKC phosphorylation site. This result was further confirmed in CHO cells stably expressing full-length MOPr. Mutating the PKC-phosphorylation site to Ala did not affect the receptor-ligand binding and receptor-G protein coupling. However, this mutation inhibited PMA-induced, but not DAMGO-induced, decrease of receptor-G protein coupling. In all, these results indicated that PKC phosphorylates MOPr C-terminus, and induces the receptor desensitization at G protein coupling level. Previous research of our lab has identified an intracellular protein, histidine triad nucleotide binding protein 1 (HINT1), interacts with MOPr and modulates receptor functions. Although HINT1 inhibits PMA-induced MOPr phosphorylation, which suggests that HINT1 may regulate MOPr function through a PKC-related manner, the molecular mechanism underlying the regulation of MOPr by HINT1 is still unknown. It is suspected that HINT1-nucleotide interaction is crucial for HINT1 biological functions, and interrupting the HINT1-nucleotide interaction will provide an opportunity to reveal the mechanism for HINT1 regulation of MOPr. Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography studies were performed to reveal the structure requirement of HINT1-nucleotide interaction. Fluorescence titration and isothermal titration calorimetry (ITC) assays further confirmed and quantified the HINT1-nucleotide interaction. Moreover, the interaction of HINT1 with AICAR, a novel non-phosphate nucleotide analogue, was also investigated. Structure information and methodologies achieved in this research provide a basis for future research aimed at revealing the mechanism of HINT1 function.
    • Phenylpropyloxyethylamines: Opioids lacking a tyrosine mimetic

      Stavitskaya, Lidiya; Coop, Andrew (2011)
      The mu opioid agonist morphine is the standard for severe pain management. Despite the ability of morphine to treat severe pain, there are significant side effects which often cause undermedication in clinical settings. Such effects are respiratory depression, tolerance, constipation, and dependence. Accordingly, investigation of novel classes of opioid analgesics would provide great therapeutic benefits. 14-Phenylpropyloxymorphinans are agonists that exhibit extreme potency at mu receptors, suggesting that the 14-phenylpropyloxy group has a major effect on receptor binding and is responsible for the dramatic increase in potency. Our hypothesis is that both a basic amine and a phenylpropyloxy group alone are required for opioid activity, and the aromatic A-ring, that was historically considered essential, is not required. By removing the A-ring, this allows the skeleton to adopt an alternate binding mode with the receptor, thereby potentially causing alternate receptor trafficking events and post-receptor mechanisms, all of which are involved in the development of tolerance. During initial studies, a conformationally sampled pharmacophore approach was utilized to confirm that the aromatic moiety in the novel series does not mimic the A-ring. In order to further substantiate our hypothesis, a series of phenylpropyloxyethylamines and cinnamyloxyethylamines were synthesized, and analyzed for opioid receptor binding affinity. Opioid binding studies showed that the optimal N-substituent is the N-phenethyl, specifically analog 2-(cinnamyloxy)-N-methyl-N-phenethylethanamine which has an affinity of 1680 nM for mu opioid receptors. Subsequently, rings B, C, and D from the morphine skeleton were systematically re-introduced as ring-constrained analogs. Binding studies showed that the B-ring analog containing a N,N-dimethyl substituent produced the highest affinity of 2340 nM, while the C- and D-ring analogs were fully inactive. Furthermore, by combining the B-ring with the optimal N-substituent, phenethyl, we were able to achieve 1640 nM affinity at mu. Moreover, upon introduction of an indole group into the C-ring analog, N,N-dimethyl-1-(3-(3-phenylpropoxy)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)methanamine, the affinity was increased to 1110 nM, which represents a viable lead compound for optimization studies.
    • 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.