Dr' Mine Yarim September' 2002

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

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• 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
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Oatps/OATPs rat/mouse Oatps human OATPs
SLC21A gene family of solute carriers (http//ww
w.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 steroid conjugates,
  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).

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Oatp3 (Slc21a7)

• rat retina, intestine
• 670 amino acid

Organic anion
Bile salts
Hormons and their conjugates
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 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).
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• 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.

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Create a 3D database (Sybyl 6.8, Tripos
Associates)
CoMFA steps
Calculate charges for each of compounds (Gasteige
r-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
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• One of the most critical adjustable parameters in
  CoMFA is the relative 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.
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Result R2 0.749
Alignment of the compounds having similar
rings.
Alignment of the all compounds (using MOE)
Estradiol-17?-glucuronide (Neutral)
Log 1/Km CoMFA PLS results
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 Glycochenodeoxycho
late Glycocholate Glycodeoxycholate Glycoursudeoxy
cholate Taurochenodeoxycholate Taurocholate Taurod
eoxycholate 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)
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• Red Negative charge
• Blue Positive charge
• Green Bulky group
• YellowLess bulky group

• The negative CoMFA region is localized in 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 a positive effect on carrier affinity.
• Estronsulfate and DHEAS with a negatively charged
  sulfate group in positive CoMFA region leads to a
  dramatic decrease in affinity.

Estronsulfate Km 268.0 ?M
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• 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?

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• 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, University Hospital,
Zürich, Switzerland