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Dr. Mine Yarim September. 2002

A COMPARATIVE MOLECULAR FIELD ANALYSIS OF ORGANIC ANION TRANSPORTING POLYPEPTIDE 3 (Oatp3) SUBSTRATES Dr. Mine Yarim September. 2002 Overview Introduction Methods and Results Outlook O rganic anion transporting polypeptides (Oatps/OATPs)

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Dr. Mine Yarim September. 2002

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  1. A COMPARATIVE MOLECULAR FIELD ANALYSIS OFORGANIC ANION TRANSPORTING POLYPEPTIDE 3 (Oatp3) SUBSTRATES Dr.Mine Yarim September. 2002

  2. Overview • Introduction • Methods and Results • Outlook

  3. Organic anion transporting polypeptides (Oatps/OATPs) • Organic cation transporters (OCTs) • Organic anion transporters (OATs) Important carrier families for uptake of drugs and endogenous compounds in liver and kidney

  4. Oatps/OATPs rat/mouse: Oatps human: OATPs SLC21A gene family of solute carriers (http://www.gene.ucl.ac.uk/nomenclature) • 80-90 kDa proteins with 12 transmembrane domains • Expressed in multiple organs • Some family members are preferentially localized in liver and • brain [1-3] • Broad substrate specifity • (bile salts, steroids and steroidconjugates, thyroid hormones, • anionic peptides and numerous drugs) • [1]. Meier, P.J., et. al, Hepatology 26, 1667 (1997). • [2]. Kullack-Ublick, G.A.,et. al, Semin Liver Dis 20, 273 (2000). • [3]. Kullack-Ublick, G.A., et. al, Gastroentrology 120 (2001).

  5. Oatp3 (Slc21a7) • rat retina, intestine • 670 aminoacid Organic anion Bile salts Hormons and their conjugates

  6. Oatp3 (Slc21a7) Substrates Km* (M) Cell Culture Lit.No. Cholate 8.8 Madin-Darby Canine Kidney[4] Glycocholate 15.4 MDCK [4] Glycodeoxycholate 4.3 MDCK [4] Glycochenodeoxycholate 5.6 MDCK [4] Glycoursodeoxycholate 5.3 MDCK [4] Taurocholate 20.9 (18) MDCK (Xenopus laevis) [4] [5] Taurodeoxycholate 5.8 MDCK [4] Taurochenodeoxycholate 7 MDCK [4] Tauroursodeoxycholate 6.6 MDCK [4] Dihydroepiandrosterone sulfate 162 Xenopus laevis [6] Estradiol-17-gulucronide 39 Xenopus laevis [6] Estrone-3-sulfate 268 Xenopus laevis [6] Prostaglandine E2 235 Xenopus laevis [6] Trijodothyronine (T3) 7 Xenopus laevis [6] Thyroxine (T4) 5 Xenopus laevis [6] BSP-Bromosulfophtalein 8.3 Xenopus laevis [6] * Km values (Michaelis constant) is a measure of the affinity of the enzyme for its substrate. Km is the substrate concentration at which the reaction velocity is half maximal. If an enzyme has a small value of Km, it achieves maximal catalytic efficiency at low substrate concentrations. [4]. Walter, H.C., et. al., Am. J. Physiology 279, G 1188 (2000). [5]. Abe, T., et. al., J. Biol. Chem. 273, 22395 (1998). [6]. Cattori, V., et. al., Pflügers Arch. – Eur. J. Physiol. 443, 188 (2001).

  7. Oatp/OATP family 3D structures are not known and nothing is known about the spatial requirements for binding to and transport of many different substrates. 3D-QSAR models may be useful to define basic requirements for binding and for future predictions of putative substrates and inhibitors of this family.

  8. CoMFA steps Create a 3D database (Sybyl 6.8, Tripos Associates) Calculate charges for each of compounds (Gasteiger-Hückel) Minimize the structure (MMFF94 force field) Alignment Calculate the steric and electrostatic field energies (+1 charged sp3 C probe atom in (2.0 Å) grid) (Steric and electrostatic contributions were truncated to a value of 30 kcal/mol) Do regression analyses (partial-least squares (PLS)) Perform using full cross-validation (leave one-out method) (To minimize the influence of noisy columns, all cross-validated analyses were performed at a minimum  (column filter) of 2.0 kcal/mol) r2 value (q2) Contour maps

  9. One of the most critical adjustable parameters in CoMFA is therelative alignment of all compounds to each other so that they have a comparable conformation and a similar spatial orientation. • In this study, we previously have tried alignment of charged substrates with similar structures. • To the alignment of the chemically diverse compounds, we selected cholate as a template. • Compounds were studied in a pair-wise comparison using the "flexible alignment" approach implemented by Molecular Operating Environment (MOE) suite [7]. • The following chemical features were selected during the flexible alignment search: Molecular volume, H-bond acceptor, H-bond donor, acidic and basic function and atomic charges. For each tested compound, conformers with the best fitness score and the lowest energy level (calculated in MOE) were selected and analyzed with CoMFA in SYBYL. [7]. Molecular Operating Environment (MOE 2002.03), Chemical Computing Group, Inc, 1255 University St., Suite 1600, Montreal, Quebec, Canada, H3B 3X3.

  10. Result R2: 0.749 Alignment of the compounds having similar rings. Alignment of the all compounds (using MOE) Log 1/Km CoMFA PLS results Estradiol-17-glucuronide (Neutral) R2: 0.782 PGEI R2: 0.748 PGEI R2: 0.748 PGEII R2: 0.745 PGEIII R2: 0.740 PGEIV R2: 0.745 PGEV R2: 0.737 Cholate DHEAS Estron-3-sulfate Glycochenodeoxycholate Glycocholate Glycodeoxycholate Glycoursudeoxycholate Taurochenodeoxycholate Taurocholate Taurodeoxycholate Tauroursodeoxycholate (Charged molecules) R2: 0.752 T4-I R2: 0.563 T4-II R2: 0.581 T4-III R2: 0.573 T4-IV R2: 0.602 T4-V R2: 0.484 T4-VIII R2: 0.605 T4-IX R2: 0.584 T4-X R2: 0.594 ProstaglandinE2 (Charged) T4-VIII R2: 0.605 Thyroxine (T4) (Amphotheric) T3-I R2: 0.682 T3-II R2: 0.682 T3-III R2: 0.681 T3-IV R2: 0.697 T3-V R2: 0.667 Triiodohyronine(T3) (Amphotheric) T3-IV R2: 0.697 BSP-I R2: 0.749 BSP-I R2: 0.749 BSP-II R2: 0.731 BSP-III R2: 0.730 BSP-IV R2: 0.729 BSP-V R2: 0.721 BSP-VI R2: 0.731 BSP-VIII R2: 0.735 BSP (Dianionic)

  11. Red: Negative charge Blue: Positive charge Green: Bulky group Yellow:Less bulky group • The negative CoMFA region is localizedin the space where most substrates contain a negatively charged group, either a carboxylate or a sulfate moiety. Glycodeoxycholate Km: 4.3 M • The introduction of steric bulk at the side chain can have apositive effect on carrier affinity. • Estronsulfate and DHEAS with a negatively charged sulfate group in positive CoMFA regionleads to a dramatic decrease in affinity. Estronsulfate Km: 268.0 M

  12. Outlook • Experiments: proposal of test set • Using by CoMFA model may predict Km value of new • compounds • Next subtype of this family? • Next transporting family?

  13. Acknowledgements • Gerd Folkers Institute of Pharmaceutical Sciences, • ETH Zürich, Switzerland • Stefano Moro Dept. of Pharm. Sci., University of Padova, Italy • (Guest Professor in ETH) • Peter Meier-Abt • Bruno Hagenbuch • Robert Huber • Flavia Pizzagalli Dept. of Internal Medicine, UniversityHospital, Zürich, Switzerland

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