SUBSTRATE DEPENDENT ALTERATIONS OF ORGANIC ANION TRANSPORTING ...

198 Pages·2011·14.42 MB·English

by  Megan Roth

SUBSTRATE DEPENDENT ALTERATIONS OF ORGANIC ANION
TRANSPORTING POLYPEPTIDE 1B3 (OATP1B3)
By
Megan E Roth

Submitted to the graduate degree program in Pharmacology, Toxicology and Therapeutics and
the Graduate Faculty of the University of Kansas Medical Center in partial fulfillment of the
requirements for the degree of Doctor of Philosophy.

Dissertation Committee:

________________________________
Bruno Hagenbuch, Ph.D., Chair

________________________________
Partha Krishnamurthy, Ph.D.

________________________________
Gregory A. Reed, Ph.D.

________________________________
Barbara N. Timmermann, Ph.D.

________________________________
Bao-Ting Zhu, Ph.D.

Date Defended: September 23, 2011
The Dissertation Committee for Megan E Roth certifies that this is the approved version of the
following dissertation:

SUBSTRATE DEPENDENT ALTERATIONS OF ORGANIC ANION
TRANSPORTING POLYPEPTIDE 1B3 (OATP1B3)

____________________________
Bruno Hagenbuch, Ph.D., Chair

Date approved: September 23, 2011

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ABSTRACT

Organic anion-transporting polypeptides (OATPs) are multispecific transporters that
mediate the uptake of numerous drugs and xenobiotics into cells. Alterations in the function of
the liver-specific OATP1B1 and OATP1B3 have been shown to affect the disposition of drugs
throughout the body. It has been proposed that new drug candidates should be screened for
possible OATP inhibition using a prototypical substrate such as estradiol-17β-glucuronide.
However, there is evidence that OATPs may have multiple binding sites, and therefore screening
with a single compound may be ineffective. Therefore, I tested the hypothesis that OATP1B3 has
multiple overlapping but distinct binding sites, which are affected in substrate-dependent ways.
This hypothesis was tested via two specific aims: 1) to identify and characterize substrate-
dependent effects of plant compounds on OATP1B3-mediated transport, and 2) to identify
regions of OATP1B3 involved in the binding and/or translocation of individual model substrates.

In the first specific aim, interacting compounds were identified by screening a library of
plant compounds for inhibition or stimulation of OATP-mediated uptake of two model
substrates. Completion of this specific aim identified two structurally similar compounds that
produce substrate-dependent effects on OATP1B3-mediated transport. These compounds
stimulate transport of estrone-3-sulfate by increasing substrate affinity. However, the compounds
either inhibit or have no effect on the uptake of estradiol-17β-glucuronide. These results
demonstrate that estrone-3-sulfate and estradiol-17β-glucuronide have distinct binding sites on
OATP1B3.

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In specific aim two, thirty-three amino acids in the first transmembrane domain and
extracellular loop of OATP1B3 were individually mutated to cysteines, and I determined the
effect of these mutations on the transport of estradiol-17β-glucuronide and estrone-3-sulfate.
Five of the cysteine-substituted OATP1B3 mutants produced different effects on transporter
function depending upon the substrate tested. These results suggest that this region of OATP1B3
is involved in the recognition and translocation of individual model substrates.

This dissertation demonstrates that OATP1B3 has distinct binding sites for estradiol-17β-
glucuronide and estrone-3-sulfate. Furthermore, it shows that transport of these two model
substrates is affected in different ways by the same compounds. This knowledge can be used to
improve screening of drug candidates to prevent adverse drug-drug interactions prior to the
occurrence of adverse events.

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Acknowledgements

This work was supported by National Institutes of Health National Institute of General Medical
Sciences Grant R01-GM077336, the National Institute of Health National Center for Research
Resources Grant P20-RR021940, the National Institute of Health Toxicology Training Grant
T32-ES07079, and the KUMC Biomedical Research Training Program Grant.

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Table of Contents in Brief

Acceptance page …………………………………………………………….……………….… ii
Abstract …………………………………………………………………….………………….. iii
Acknowledgements …………………………..…………………………….………………….. iv
Table of Contents in Brief ………………………………………………….………………….. vi
Table of Contents Expanded ……………………………………………….…………………. vii
List of Tables ……………………………………………………………….………………… xii
List of Figures …………………………………………………………….………………….. xiii
List of Abbreviations …………………………………………………….…………………… xv
List of Appendices …………………………………………………………………………. xviii

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Table of Contents Expanded

Chapter 1
Background and Significance

1.1 Drug Absorption and Distribution …………………………………………….………… 1
1.2 Transporters Involved in Drug Absorption, Distribution, and Elimination …………...… 2
1.3 The Organic Anion Transporting Polypeptide (OATP) Superfamily …………………… 3
1.3.1 Nomenclature …………………………………………………...……………….. 3
1.3.2 Tissue Distribution ……………………………………………….……………… 4
1.3.2a Normal Expression ………………………………………………………. 4
1.3.2b Dysregulated Expression …………………………………….………….. 8
1.3.2c Physiological Consequences of Expression ………………….…………. 8
1.3.3 Substrate Specificity …………………………………………………………… 13
1.3.4 Mechanism of Transport ……………………………………………….………. 27
1.3.5 Structure of OATPs ……………………………………………………………. 29
1.3.6 Polymorphisms ………………………………………………………………… 32
1.4 Interactions with Drug Transporters: Drugs, Foods, and Small Molecules …….……… 34
1.4.1 Adverse drug-drug interactions ………………………………………………… 34
1.4.2 Adverse drug-food interactions …………………………………..…………….. 36
1.4.3 Beneficial drug interactions: targeted treatment ……………….………………. 36
1.5 Specific Aims of this Dissertation ………………………………...…………………… 37

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1.5.1 Specific Aim 1: Identify and characterize substrate-dependent effects
of plant compounds on OATP-mediated transport………………..……….…… 39
1.5.1a: Interaction of green tea catechins with organic
anion transporting polypeptides ………………..………………………. 39
1.5.1b Isolation of modulators of organic anion transporting
polypeptides 1B1 and 1B3 from Rollinia emarginata
Schlecht (Annonaceae) …………………………...…………….……… 39
1.5.2 Specific Aim 2: Identify regions of OATP1B3 involved in the binding
and/or translocation of individual model substrates ……..………….….……… 40

Chapter 2
Experimental Materials and Methods

2.1 Materials ……………………………………………………………………………….. 41
2.2 Plant Compound Extraction and Isolation …………………………...………………… 42
2.3 Plant Compound Identification ………………………………………..……………….. 43
2.4 Site-directed Mutagenesis ……………………………………………………………… 44
2.5 Cell Culture …………………………………………………………….………………. 45
2.6 OATP Expression ………………………………………………………..…………….. 45
2.7 Transport Assays …………………………………………………………..…………… 46
2.8 Cell Surface Biotinylation and Immunoblot Analysis …………………………………. 48
2.9 Methanethiosulfonate and Maleimide Accessibility Reactions ………….…………….. 48

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2.10 Calculations and Statistics ……………………………………………….…………….. 49

Chapter 3
Interactions of Green Tea Catechins with Organic Anion-Transporting Polypeptides

3.1 Abstract ……………………………………………………………………………..….. 50
3.2 Introduction ……………………………………………………………………….….… 51
3.3 Results ………………………………………………………………………………….. 53
3.3.1 Characterization of OATP1A2 and OATP2B1 Expression Systems ………….. 53
3.3.2 Effect of Green Tea Extract and Catechins on OATP-Mediated
Uptake of Estrone-3-Sulfate …………………………………………………… 54
3.3.3 Substrate-Dependent Effect of ECG and EGCG on
OATP1B3-Mediated Uptake …………………………………………………... 59
3.3.4 OATP-Mediated Uptake of ECG and EGCG …………………………………. 64
3.4 Discussion …………………………………………………………………………...… 69

Chapter 4
Isolation of Modulators of the Liver Specific Organic Anion Transporting Polypeptides (OATPs)
1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae)

4.1 Abstract ……………….………………………………………………………………. 74

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4.2 Introduction …………………………………………………………………………….. 75
4.3 Results ………………………………………………………………………………….. 78
4.3.1 Identification of Compounds with Modulating Effects
on OATP1B1 and OATP1B3 ………………………………………………….. 78
4.3.2 Ursolic Acid, Oleanolic Acid, and 8-trans-p-coumaroyloxy-
α-terpineol Selectively Inhibit OATP-Mediated Transport
of Estradiol-17β-glucuronide ……………………………………...…………… 82
4.3.3 Quercetin 3-Ο-α-L-arabinopyranosyl (1→2) α-L-rhamnopyranoside
has Concentration-dependent Effects on OATP1B1- and
OAPT1B3-mediated Uptake ………………………………………………..….. 88
4.3.4 Quercetin 3-Ο-α-L-arabinopyranosyl (1→2) α-L-rhamnopyranoside
Decreases OATP1B3’s Maximal Transport Rate for Estradiol-17β-
glucuronide and Estrone-3-sulfate, and Increases its Affinity
for Estrone-3-sulfate …………………………………………………………… 94
4.4 Discussion …………………………………………………..………………..……….. 102

Chapter 5
Cysteine Scanning Mutagenesis on Transmembrane Domain 1 of the Liver-Specific Organic
Anion Transporting Polypeptide 1B3 (OATP1B3)

5.1 Abstract ……………………………………………………………………………….. 107
5.2 Introduction ………………………………………………………..………………….. 108

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