Publications by authors named "Peter Swaan"

106 Publications

S-acylation status of bile acid transporter hASBT regulates its function, metabolic stability, membrane expression, and phosphorylation state.

Biochim Biophys Acta Biomembr 2021 02 13;1863(2):183510. Epub 2020 Nov 13.

Department of Pharmaceutical Sciences, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA. Electronic address:

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (J) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.
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http://dx.doi.org/10.1016/j.bbamem.2020.183510DOI Listing
February 2021

Quantification of common and planar bile acids in tissues and cultured cells.

J Lipid Res 2020 11 22;61(11):1524-1535. Epub 2020 Jul 22.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Baltimore, MD, USA

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, , in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.
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http://dx.doi.org/10.1194/jlr.D120000726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604731PMC
November 2020

Farewell Message from the Editor-in-Chief.

Authors:
Peter W Swaan

Pharm Res 2020 07 8;37(7):135. Epub 2020 Jul 8.

Department of Pharmaceutical Sciences, University of Maryland, 20 Penn Street, Baltimore, Maryland, 21201, USA.

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http://dx.doi.org/10.1007/s11095-020-02870-4DOI Listing
July 2020

Trends in Research and Graduate Programs in Schools and Colleges of Pharmacy, Part 1: Programs.

Am J Pharm Educ 2020 05;84(5):7643

University of Maryland School of Pharmacy, Baltimore, Maryland.

To examine the landscape of research and graduate education nationally and within schools and colleges of pharmacy. This report is part 1 of a three-part series and focuses on graduate programs' research funding and science faculty composition and diversity. Between FY2008 and FY2017, the number of full-time faculty members in schools and colleges of pharmacy increased 36%. The number of pharmacy schools with National Institutes of Health (NIH) awards increased by 15%, while NIH grants per faculty principal investigator (PI) increased by 31%. However, unadjusted for inflation, the mean NIH dollar amount per-faculty member PI increased just 14% and the mean NIH dollar amount per-school declined 7%, indicating that number of funded faculty outpaced dollars available. Proportionately, the percentage of science faculty members at pharmacy schools decreased from 47% to 43%. Only 15 public, research-intensive schools and colleges of pharmacy received more than half of the combined FY2017 NIH funding and total funding, while all other public and private schools and colleges of pharmacy shared the remaining funds. Interdisciplinary programs are developing slowly, and may help to diversify and increase future funding. Proportions of tenured and tenure-track positions are declining, but biological sciences and social and administrative sciences disciplines are growing and women faculty are making significant gains in these fields and at the assistant professor rank. Research-intensive schools and colleges of pharmacy are best-positioned to lead the academy to reframe graduate education to build interdisciplinary team skills and attract more diverse funding and science faculty members.
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http://dx.doi.org/10.5688/ajpe7643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298211PMC
May 2020

Trends in Research and Graduate Affairs in Schools and Colleges of Pharmacy, Part 2: Students.

Am J Pharm Educ 2020 05;84(5):7642

University of Maryland School of Pharmacy, Baltimore, Maryland.

To examine the landscape of research and graduate affairs nationally and within schools and colleges of pharmacy. This report, part 2 of a three-part series, focuses on characteristics of full-time PhD enrollees and graduates in schools and colleges of pharmacy, and career planning and preparation in graduate programs. Despite a 41% increase in funding awarded by the National Institutes of Health (NIH) to schools and colleges of pharmacy over the last 10 years, NIH funding per principal investigator only increased 14% and graduate student enrollment increased just 6% during the period. However, there was a 15% increase in PhD degrees conferred in the 10-year period, which is evidence that degree completion time decreased. The number of female graduates from pharmacy schools consistently increased, and outpaced growth in the number of male graduates by more than 10%. Most graduate programs do not include training for industry-specific skills, abilities, and experiences to better prepare graduates for nonacademic careers, although national programs have been recognized as vital to graduate student career preparation. Graduate biomedical science programs and faculty members must recognize that academia is an "alternative" career choice for their trainees, and provide job skills training to support the majority of nonacademic career choices, without compromising the rigorous training in basic biomedical disciplines.
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http://dx.doi.org/10.5688/ajpe7642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298219PMC
May 2020

Trends in Research and Graduate Affairs in Schools and Colleges of Pharmacy, Part 3: Underrepresented Minorities.

Am J Pharm Educ 2020 05;84(5):7641

University of Maryland School of Pharmacy, Baltimore, Maryland.

To examine the landscape of research and graduate affairs nationally and within schools and colleges of pharmacy. This report, part 3 of a three-part series, focuses on underrepresented minority (URM) faculty members and students, with a focus on recruitment and retention. There has been a substantial increase in recruitment of Asian faculty members by schools of pharmacy over the last 10 years, but there has been only minimal changes in the numbers of Black and Hispanic faculty numbers, which reflects the challenges in recruitment and retention of URM faculty members. Consistently low enrollment of Black and Hispanic graduate students over a 10-year period demonstrates that pharmacy schools could improve their stated diversity initiatives and goals. Despite an overall increase in PhDs conferred over the last 10 years, international students continue to receive the majority of degrees conferred. Graduation rates of Black and Hispanic students have remained low, suggesting that continued and sustained efforts are needed to recruit, support, and graduate URM students. Pharmacy schools must make a focused investment and effort toward increasing the diversity of their graduate enrollees by modeling their recruitment, enrollment, and retention strategies after national programs and best practices. Because there is a direct link between the number of faculty role models and the recruitment of students, pharmacy schools must enhance the recruitment, retention, and success of URM faculty members. Further, pharmacy schools should provide inclusion training to encourage better communication with URM advisees.
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http://dx.doi.org/10.5688/ajpe7641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298212PMC
May 2020

Mechanistic Insights of Phenobarbital-Mediated Activation of Human but Not Mouse Pregnane X Receptor.

Mol Pharmacol 2019 09 10;96(3):345-354. Epub 2019 Jul 10.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (L.L., M.A.W., Z.L., B.M., P.W.S., H.W.); and BioIVT, Halethorpe, Maryland (S.H.)

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (K = 1.42 × 10). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.
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http://dx.doi.org/10.1124/mol.119.116616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701513PMC
September 2019

Tyrosine Phosphorylation Regulates Plasma Membrane Expression and Stability of the Human Bile Acid Transporter ASBT (102).

Mol Pharm 2019 08 8;16(8):3569-3576. Epub 2019 Jul 8.

Department of Pharmaceutical Sciences , University of Maryland , 20 Penn Street , Baltimore , Maryland 21201 , United States.

The human apical sodium-dependent bile acid transporter (hASBT; 102) is responsible for the reclamation of bile acids from the intestinal lumen, providing a primary mechanism for bile acid and cholesterol homeostasis. However, the regulation of hASBT at the post-translational level is not well understood. In the present study, we investigated the role of Src family kinases (SFKs) and protein tyrosine phosphatases (PTPs) in the regulation of surface expression and function of hASBT. Inhibition of Src family kinases, via treatment with PP2, significantly reduced hASBT function, while the inhibition of PTPs by activated orthovanadate significantly induced function. Src family kinase inhibition by PP2 was associated with a concomitant decrease in maximum transport velocity () correlated with a decrease in hASBT surface expression. Interestingly, PP2-mediated suppression of hASBT protein expression was rescued by the proteasome inhibitor MG132, suggesting that dephosphorylation impacts protein stability with the subsequent proteasome-dependent degradation of hASBT. Consequently, single-point mutations were introduced at five intracellular tyrosine residues: Y148F, Y216F, Y308F, Y311F, and Y337F. Although all mutants had significantly altered hASBT function without changes in total cellular expression, sequential tyrosine mutations at the five residues above rendered hASBT nonfunctional with diminished protein expression. Furthermore, orthovanadate-induced transport activity of single-point tyrosine mutants suggested a role for multiple tyrosine residues in the regulation of hASBT function and membrane expression. Overall, our data confirms that tyrosine phosphorylation mediated by Src family kinases (SFKs), in particular, regulates surface expression, function, and stability of hASBT.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00426DOI Listing
August 2019

Set of Highly Stable Amine- and Carboxylate-Terminated Dendronized Au Nanoparticles with Dense Coating and Nontoxic Mixed-Dendronized Form.

Langmuir 2019 03 18;35(9):3391-3403. Epub 2019 Feb 18.

Department of Chemistry and Biochemistry , University of Maryland Baltimore County (UMBC) , Baltimore , Maryland 21250 , United States.

The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug delivery platform is described herein. The AuNP-based platform is composed of three complementary parts: (i) a 15 nm AuNP core, (ii) a heterofunctional thioctic acid-terminated tetraethylene glycol spacer, and (iii) a third-generation PPI dendron with a unique protonation profile and diverse end-group functionalization that allows for further derivatization. The prepared dendronized AuNPs are able to withstand several rounds of lyophilization cycles with no sign of aggregation, are stable in phosphate-buffered saline and Hanks' buffer as well as in serum, and are resistant to degradation by glutathione exchange reactions. This nanocarrier platform displays a dense coating, with >1400 dendrons/AuNPs, which will enable very high payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against the MCF-7 breast cancer cell line from a NP concentration of 1 nM, the mixed monolayer AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of toxicity at concentrations as high as 15 nM, similar to the carboxylate-terminated AuNPs. The described dendronized AuNPs address the current practical need for a stable NP-based drug delivery platform which is scalable and easily conjugable, has long-term stability in solution, and can be conveniently formulated as a powder and redispersed in desired buffer or serum.
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http://dx.doi.org/10.1021/acs.langmuir.8b03196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499480PMC
March 2019

Molecular Modeling of Drug-Transporter Interactions-An International Transporter Consortium Perspective.

Clin Pharmacol Ther 2018 11 30;104(5):818-835. Epub 2018 Aug 30.

Department of Pharmacy, Uppsala University, Uppsala, Sweden.

Membrane transporters play diverse roles in the pharmacokinetics and pharmacodynamics of small-molecule drugs. Understanding the mechanisms of drug-transporter interactions at the molecular level is, therefore, essential for the design of drugs with optimal therapeutic effects. This white paper examines recent progress, applications, and challenges of molecular modeling of membrane transporters, including modeling techniques that are centered on the structures of transporter ligands, and those focusing on the structures of the transporters. The goals of this article are to illustrate current best practices and future opportunities in using molecular modeling techniques to understand and predict transporter-mediated effects on drug disposition and efficacy.Membrane transporters from the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies regulate the cellular uptake, efflux, and homeostasis of many essential nutrients and significantly impact the pharmacokinetics of drugs; further, they may provide targets for novel therapeutics as well as facilitate prodrug approaches. Because of their often broad substrate selectivity they are also implicated in many undesirable and sometimes life-threatening drug-drug interactions (DDIs)..
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http://dx.doi.org/10.1002/cpt.1174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197929PMC
November 2018

Post-translational modifications of transporters.

Pharmacol Ther 2018 12 30;192:88-99. Epub 2018 Jun 30.

Department of Pharmaceutical Sciences, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA. Electronic address:

Drug transporter proteins are critical to the distribution of a wide range of endogenous compounds and xenobiotics such as hormones, bile acids, peptides, lipids, sugars, and drugs. There are two classes of drug transporters- the solute carrier (SLC) transporters and ATP-binding cassette (ABC) transporters -which predominantly differ in the energy source utilized to transport substrates across a membrane barrier. Despite their hydrophobic nature and residence in the membrane bilayer, drug transporters have dynamic structures and adopt many conformations during the translocation process. Whereas there is significant literature evidence for the substrate specificity and structure-function relationship for clinically relevant drug transporters proteins, there is less of an understanding in the regulatory mechanisms that contribute to the functional expression of these proteins. Post-translational modifications have been shown to modulate drug transporter functional expression via a wide range of molecular mechanisms. These modifications commonly occur through the addition of a functional group (e.g. phosphorylation), a small protein (e.g. ubiquitination), sugar chains (e.g. glycosylation), or lipids (e.g. palmitoylation) on solvent accessible amino acid residues. These covalent additions often occur as a result of a signaling cascade and may be reversible depending on the type of modification and the intended fate of the signaling event. Here, we review the significant role in which post-translational modifications contribute to the dynamic regulation and functional consequences of SLC and ABC drug transporters and highlight recent progress in understanding their roles in transporter structure, function, and regulation.
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http://dx.doi.org/10.1016/j.pharmthera.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6263853PMC
December 2018

Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.

Biochim Biophys Acta Biomembr 2018 Mar 1;1860(3):645-653. Epub 2017 Dec 1.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA. Electronic address:

The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.
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http://dx.doi.org/10.1016/j.bbamem.2017.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780253PMC
March 2018

Planar bile acids in health and disease.

Biochim Biophys Acta Biomembr 2017 Nov 6;1859(11):2269-2276. Epub 2017 Sep 6.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA. Electronic address:

Bile acids are the amphipathic primary end-products of cholesterol metabolism that aid in digestion as well as participate in signal transduction in several hepatic and enteric pathways. Despite the reputation of bile acids as signaling molecules implicated in disease states such as cancer and diabetes, there remain numerous bile acid species that are weakly characterized in either physiological or pathological conditions. This review presents one such group: the flat or planar bile acids, a set of bile acids found in humans during infancy and occurring again during certain diseases. As their name implies, these molecules are structurally distinct from the typical human bile acids, retaining the planar structure of their cholesterol predecessor instead of bending or twisting at the A ring. This review defines these species of bile acids in detail and describes their presence in infancy, gestation, and in disease. The large gaps in research regarding the flat bile acids are highlighted and all available experimental knowledge collected as far as 60years ago is summarized. Further, the potential for these molecules as endogenous biomarkers of liver disease and injury is discussed. Finally, the flat bile salts found in humans are compared to the ancestral and evolutionary older bile salts, which similarly have a flat steroidal structure, as mechanisms of flat bile acid biosynthesis are explored.
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http://dx.doi.org/10.1016/j.bbamem.2017.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734676PMC
November 2017

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

Mol Pharmacol 2017 07 25;92(1):75-87. Epub 2017 Apr 25.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (B.M., L.L., M.A.W., D.L., J.W.J., M.A.K., P.W.S., H.W.); and Bioreclamation In Vitro Technologies, Halethorpe, Maryland (S.H.)

The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of the CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. 1-(2-chlorophenyl)--methyl--(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a typical peripheral benzodiazepine receptor ligand, has been established as a potent hCAR deactivator in immortalized cells; whether it inhibits hCAR activity under physiologically relevant conditions remains unclear. Here, we investigated the effects of PK11195 on hCAR in metabolically competent human primary hepatocytes (HPH) and HepaRG cells. We show that although PK11195 antagonizes hCAR in HepG2 cells, it induces the expression of CYP2B6 and CYP3A4, targets of hCAR and the pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockout HepaRG cells. Utilizing a HPH-HepG2 coculture model, we demonstrate that inclusion of HPH converts PK11195 from an antagonist to an agonist of hCAR, and such conversion was attenuated by potent CYP3A4 inhibitor ketoconazole. Metabolically, we show that the -desmethyl metabolite is responsible for PK11195-mediated hCAR activation by facilitating hCAR interaction with coactivators and enhancing hCAR nuclear translocation in HPHs. Structure-activity analysis revealed that -demethylation alters the interaction of PK11195 with the binding pocket of hCAR to favor activation. Together, these results indicate that removal of a methyl group switches PK11195 from a potent antagonist of hCAR to an agonist in HPH and highlights the importance of physiologically relevant metabolism when attempting to define the biologic action of small molecules.
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http://dx.doi.org/10.1124/mol.117.108621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452073PMC
July 2017

Identification of novel MRP3 inhibitors based on computational models and validation using an in vitro membrane vesicle assay.

Eur J Pharm Sci 2017 May 24;103:52-59. Epub 2017 Feb 24.

Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Introduction: Multidrug resistance-associated protein 3 (MRP3), an efflux transporter on the hepatic basolateral membrane, may function as a compensatory mechanism to prevent the accumulation of anionic substrates (e.g., bile acids) in hepatocytes. Inhibition of MRP3 may disrupt bile acid homeostasis and is one hypothesized risk factor for the development of drug-induced liver injury (DILI). Therefore, identifying potential MRP3 inhibitors could help mitigate the occurrence of DILI.

Methods: Bayesian models were developed using MRP3 transporter inhibition data for 86 structurally diverse drugs. The compounds were split into training and test sets of 57 and 29 compounds, respectively, and six models were generated based on distinct inhibition thresholds and molecular fingerprint methods. The six Bayesian models were validated against the test set and the model with the highest accuracy was utilized for a virtual screen of 1470 FDA-approved drugs from DrugBank. Compounds that were predicted to be inhibitors were selected for in vitro validation. The ability of these compounds to inhibit MRP3 transport at a concentration of 100μM was measured in membrane vesicles derived from stably transfected MRP3-over-expressing HEK-293 cells with [H]-estradiol-17β-d-glucuronide (E17G; 10μM; 5min uptake) as the probe substrate.

Results: A predictive Bayesian model was developed with a sensitivity of 73% and specificity of 71% against the test set used to evaluate the six models. The area under the Receiver Operating Characteristic (ROC) curve was 0.710 against the test set. The final selected model was based on compounds that inhibited substrate transport by at least 50% compared to the negative control, and functional-class fingerprints (FCFP) with a circular diameter of six atoms, in addition to one-dimensional physicochemical properties. The in vitro screening of predicted inhibitors and non-inhibitors resulted in similar model performance with a sensitivity of 64% and specificity of 70%. The strongest inhibitors of MRP3-mediated E17G transport were fidaxomicin, suramin, and dronedarone. Kinetic assessment revealed that fidaxomicin was the most potent of these inhibitors (IC=1.83±0.46μM). Suramin and dronedarone exhibited IC values of 3.33±0.41 and 47.44±4.41μM, respectively.

Conclusion: Bayesian models are a useful screening approach to identify potential inhibitors of transport proteins. Novel MRP3 inhibitors were identified by virtual screening using the selected Bayesian model, and MRP3 inhibition was confirmed by an in vitro transporter inhibition assay. Information generated using this modeling approach may be valuable in predicting the potential for DILI and/or MRP3-mediated drug-drug interactions.
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http://dx.doi.org/10.1016/j.ejps.2017.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450158PMC
May 2017

Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors.

Drug Metab Dispos 2015 Nov 18;43(11):1773-80. Epub 2015 Aug 18.

Department of Pharmaceutics, University of Washington, Seattle, Washington (H.D., T.H., J.W.); Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington (R.S.F.); and Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (Y.P., P.W.S.)

Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1-3 (OCT1-3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1-3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC₅₀ = 1.4 ± 0.2 µM) over OCT1 (IC₅₀ = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1-3.
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http://dx.doi.org/10.1124/dmd.115.064824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613949PMC
November 2015

Obituary: Paul M. Bummer (1955 - 2015).

Authors:
Peter W Swaan

Pharm Res 2015 Sep;32(9):2813

University of Maryland, Baltimore, MD, USA,

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http://dx.doi.org/10.1007/s11095-015-1757-4DOI Listing
September 2015

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

Mol Pharm 2015 Jun 19;12(6):1961-9. Epub 2015 May 19.

Department of Pharmaceutical Sciences, Center for Nanobiotechnology, University of Maryland, Baltimore, 20 Penn Street, Baltimore, Maryland 21201, United States.

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.
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http://dx.doi.org/10.1021/mp500765eDOI Listing
June 2015

Toward predicting drug-induced liver injury: parallel computational approaches to identify multidrug resistance protein 4 and bile salt export pump inhibitors.

Drug Metab Dispos 2015 May 3;43(5):725-34. Epub 2015 Mar 3.

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (M.A.W., P.W.S.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., T.J.U., K.L.R.B.); Center for Human Genome Variation, Duke University Medical Center, Durham, North Carolina (T.J.U.)

Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of multidrug resistance protein 4 (MRP4), in addition to bile salt export pump (BSEP), might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed using 257 BSEP and 86 MRP4 inhibitors and noninhibitors in the training set. Models were externally validated and used to predict the affinity of compounds toward BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of nine well characterized MRP4 inhibitors and nine potent BSEP inhibitors. Bayesian models for BSEP and MRP4 inhibition/noninhibition were developed with cross-validated receiver operator curve values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements. Similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability.
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http://dx.doi.org/10.1124/dmd.114.062539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407708PMC
May 2015

Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes.

Toxicol Appl Pharmacol 2014 Aug 27;279(1):33-42. Epub 2014 May 27.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA. Electronic address:

Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH).

Methods: Ligand-based structure-activity models were used for virtual screening of the Specs database (www.specs.net) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH.

Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo.

Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis.

Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders.
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http://dx.doi.org/10.1016/j.taap.2014.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118694PMC
August 2014

Intracellular Ca2+ release mediates cationic but not anionic poly(amidoamine) (PAMAM) dendrimer-induced tight junction modulation.

Pharm Res 2014 Sep 20;31(9):2429-38. Epub 2014 Mar 20.

Department of Pharmaceutical Sciences Center for Nanomedicine and Cellular Drug Delivery, University of Maryland, Baltimore, 20 Penn Street, Health Sciences Facility II, Room 543, Baltimore, Maryland, 21201, USA.

Purpose: Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation.

Methods: Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [(14)C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability.

Results: Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers.

Conclusions: The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.
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http://dx.doi.org/10.1007/s11095-014-1338-yDOI Listing
September 2014

The role of transporters in toxicity and disease.

Drug Metab Dispos 2014 Apr;42(4):541-5

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (J.D.S); Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (P.W.S); and Department of Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim, Ridgefield, Connecticut (D.J.T.).

The significance of transporters in the disposition, metabolism, and elimination of drugs is well recognized. One gap in our knowledge is a comprehensive understanding of how drug transporters change functionality (their amount and activity) in response to disease and how disease and its inevitable pathology change transporter expression. In this issue of Drug Metabolism and Disposition a series of review and primary research articles are presented to highlight the importance of transporters in toxicity and disease. Because of the central role of the liver in drug metabolism, many of the articles in this theme issue focus on transporters in the liver and how pathology or alterations in physiology affects transporter expression. The contributing authors have also considered the role of transporters in drug interactions as well as drug-induced liver injury. Noninvasive approaches to assessing transporter function in vivo are also described. Several articles highlight important issues in oncology where toxicity must be balanced against efficacy. In total, this theme issue will provide a stepping-stone to future studies that will establish a more comprehensive understanding of transporters in disease.
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http://dx.doi.org/10.1124/dmd.114.057539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965901PMC
April 2014

Resveratrol promotes degradation of the human bile acid transporter ASBT (SLC10A2).

Biochem J 2014 Apr;459(2):301-12

*Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, U.S.A.

The sodium/bile acid co-transporter ASBT [apical sodium-dependent bile acid transporter; SLC10A2 (solute carrier family 10 member 2)] plays a key role in the enterohepatic recycling of the bile acids and indirectly contributes to cholesterol homoeostasis. ASBT inhibitors reportedly lower plasma triglyceride levels and increase HDL (high-density lipoprotein) cholesterol levels. RSV (resveratrol), a major constituent of red wine, is known to lower LDL (low-density lipoprotein) cholesterol levels, but its mechanism of action is still unclear. In the present study, we investigated the possible involvement of ASBT in RSV-mediated cholesterol-lowering effects. We demonstrate that RSV inhibits ASBT protein expression and function via a SIRT1 (sirtuin 1)-independent mechanism. The effect was specific to ASBT since other transporters involved in cholesterol homoeostasis, NTCP (SLC10A1), OSTα (SLC51A) and ABCG1 (ATP-binding cassette G1), remained unaffected. ASBT inhibition by RSV was reversed by proteasome inhibitors (MG-132 and lactacystin) and the ubiquitin inhibitor LDN57444, suggesting involvement of the ubiquitin-proteasome pathway. Immunoprecipitation revealed high levels of ubiquitinated ASBT after RSV treatment. Phosphorylation at Ser335 and Thr339 was shown previously to play a role in proteosomal degradation of rat ASBT. However, mutation at corresponding residues in rat ASBT revealed that phosphorylation does not contribute to RSV-mediated degradation of ASBT. Combined, our data indicate that RSV promotes ASBT degradation via the ubiquitin-proteasome pathway without requiring phosphorylation. We conclude that regulation of ASBT expression by RSV may have clinical relevance with regard to the observed cholesterol-lowering effects of RSV.
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http://dx.doi.org/10.1042/BJ20131428DOI Listing
April 2014

Risk factors for development of cholestatic drug-induced liver injury: inhibition of hepatic basolateral bile acid transporters multidrug resistance-associated proteins 3 and 4.

Drug Metab Dispos 2014 Apr 23;42(4):665-74. Epub 2013 Oct 23.

Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy (K.K., B.C.F., I.N., K.Y., K.L.R.B.), and Biostatistics Department, School of Public Health (P.W.St.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Pharmacy, Uppsala University, Uppsala, Sweden (I.N.); Center for Human Genome Variation, Duke University Medical Center, Durham, North Carolina (T.J.U.); and Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (P.W.Sw.).

Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 μM) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e.g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI.
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http://dx.doi.org/10.1124/dmd.113.054304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965904PMC
April 2014

Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms.

Pharm Res 2014 Feb 3;31(2):401-13. Epub 2013 Oct 3.

Center for Nanomedicine and Cellular Delivery Department of Pharmaceutical Science, University of Maryland, Baltimore, Maryland, USA.

Purpose: This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated.

Methods: Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy.

Results: Liposome uptake by Caco-2 cells was observed to be strongly size-dependent for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter.

Conclusions: The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.
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http://dx.doi.org/10.1007/s11095-013-1171-8DOI Listing
February 2014

Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.

J Biol Chem 2013 Nov 17;288(45):32394-32404. Epub 2013 Sep 17.

From the Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201. Electronic address:

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.
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http://dx.doi.org/10.1074/jbc.M113.518555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820874PMC
November 2013

Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2.

Biochemistry 2013 Jul 16;52(30):5117-24. Epub 2013 Jul 16.

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, United States.

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants having a significantly decreased transport activity, although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional, and G201 and G212 functioned at a level of <10% upon cysteine mutation. Interestingly, each of these exists along one face of the helix. Studies suggest that neither G201 nor G212 is on the substrate pathway. Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face transmembrane domain 4 (TM4) residues G179 and P175, respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in transmembrane domain 6 (TM6). It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation.
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http://dx.doi.org/10.1021/bi400028qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812428PMC
July 2013

Riboflavin-targeted polymer conjugates for breast tumor delivery.

Pharm Res 2013 Jul 9;30(7):1799-812. Epub 2013 Apr 9.

Department of Pharmaceutical Sciences Center for Nanomedicine and Cellular Drug Delivery, University of Maryland Baltimore, 20 Penn Street, Health Sciences Facility 2, Room 543, Baltimore, Maryland 21201, USA.

Purpose: In breast cancer, a significant decrease in riboflavin (RF) serum levels and increase in RF carrier protein occurs, indicating a potential role of RF in disease progression. To evaluate RF's ability to serve as a targeting agent, mitomycin C (MMC)-conjugated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and targeted to the RF internalization pathway in human breast cancer cells.

Methods: Competitive uptake studies were used to determine specificity of RF-targeted conjugates, and an MTT assay established the IC₅₀ for the conjugates. Endocytic mechanisms were investigated by confocal microscopy.

Results: Studies revealed a high-affinity endocytic mechanism for RF-specific internalization of fluorescently-labeled conjugates in both MCF-7 and SKBR-3 cells, whereas folic acid-mediated endocytosis showed high specificity only in SKBR-3 cells. MMC internalization was significantly higher following nontargeted and RF-targeted MMC-conjugate administration compared to that of free MMC. Cytotoxic analysis illustrated potent IC₅₀ values for RF-targeted MMC conjugates similar to free MMC. Maximum nuclear accumulation of MMC resulted from lysosomal release from RF-targeted and nontargeted MMC-conjugates following 6 h incubations, unlike that of free MMC seen within 10 min.

Conclusion: Targeting polymer-MMC conjugates to the RF internalization pathway in breast cancer cells enabled an increase in MMC uptake and nuclear localization, resulting in potent cytotoxic activity.
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http://dx.doi.org/10.1007/s11095-013-1024-5DOI Listing
July 2013

The solute carrier family 10 (SLC10): beyond bile acid transport.

Mol Aspects Med 2013 Apr-Jun;34(2-3):252-69

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, USA.

The solute carrier (SLC) family 10 (SLC10) comprises influx transporters of bile acids, steroidal hormones, various drugs, and several other substrates. Because the seminal transporters of this family, namely, sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2), were primarily bile acid transporters, the term "sodium bile salt cotransporting family" was used for the SLC10 family. However, this notion became obsolete with the finding of other SLC10 members that do not transport bile acids. For example, the sodium-dependent organic anion transporter (SOAT; SLC10A6) transports primarily sulfated steroids. Moreover, NTCP was shown to also transport steroids and xenobiotics, including HMG-CoA inhibitors (statins). The SLC10 family contains four additional members, namely, P3 (SLC10A3; SLC10A3), P4 (SLC10A4; SLC10A4), P5 (SLC10A5; SLC10A5) and SLC10A7 (SLC10A7), several of which were unknown or considered hypothetical until approximately a decade ago. While their substrate specificity remains undetermined, great progress has been made towards their characterization in recent years. Explicitly, SLC10A4 may participate in vesicular storage or exocytosis of neurotransmitters or mastocyte mediators, whereas SLC10A5 and SLC10A7 may be involved in solute transport and SLC10A3 may have a role as a housekeeping protein. Finally, the newly found role of bile acids in glucose and energy homeostasis, via the TGR5 receptor, sheds new light on the clinical relevance of ASBT and NTCP. The present mini-review provides a brief summary of recent progress on members of the SLC10 family.
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http://dx.doi.org/10.1016/j.mam.2012.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602841PMC
September 2013

Identification of novel breast cancer resistance protein (BCRP) inhibitors by virtual screening.

Mol Pharm 2013 Apr 21;10(4):1236-48. Epub 2013 Mar 21.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, Maryland 21201, USA.

Breast cancer resistance protein (BCRP; ABCG2) is an efflux transporter that plays an important role in multidrug resistance to antineoplastic drugs. The identification of drugs as BCRP inhibitors could aid in designing better therapeutic strategies for cancer treatment and will be critical for identifying potential drug-drug interactions. In the present study, we applied ligand-based virtual screening combined with experimental testing for the identification of novel drugs that can possibly interact with BCRP. Bayesian and pharmacophore models generated with known BCRP inhibitors were validated with an external test set. The resulting models were applied to predict new potential drug candidates from a database with more than 2000 FDA-approved drugs. Thirty-three drugs were tested in vitro for their inhibitory effects on BCRP-mediated transport of [(3)H]-mitoxantrone in MCF-7/AdrVp cells. Nineteen drugs were identified with significant inhibitory effect on BCRP transport function. The combined strategy of computational and experimental approaches in this paper has suggested potential drug candidates and thus represents an effective tool for rational identification of modulators of other proteins.
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http://dx.doi.org/10.1021/mp300547hDOI Listing
April 2013