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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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slide1

A COMPARATIVE MOLECULAR FIELD ANALYSIS OFORGANIC ANION TRANSPORTING POLYPEPTIDE 3 (Oatp3) SUBSTRATES

Dr.Mine Yarim September. 2002

slide2

Overview

  • Introduction
  • Methods and Results
  • Outlook
slide3

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

slide4

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).
slide5

Oatp3 (Slc21a7)

  • rat retina, intestine
  • 670 aminoacid

Organic anion

Bile salts

Hormons and their conjugates

slide6

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).

slide7
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.

slide8

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

slide9

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.

slide10

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)

slide11
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

slide12

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?
slide13

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