Download Lausanne 2002

Università degli Studi di Milano
Molecular docking and QSAR analysis:
a combined approach applied to FTase
inhibitors and a1a-AR antagonists
Giulio Vistoli, Alessandro Pedretti
The Farnesyltransferase
•The Farnesyltransferase (FTase) catalyzes the transfer of a farnesyl group
from farnesyl diphosphate (FPP) to a specific cysteine residue of a
substrate protein through covalent attachment.
•This post-translational modification is believed to be involved in membrane
association due to the enhanced hydrophobicity of the protein upon
farnesylation.
•This modification process has been identified in the Ras proteins that play
a crucial role in the signal transduction pathway that leads to cell division.
•Preventing the farnesylation process may be a possible approach for anticancer chemotherapy.
•Knowledge about the active site environment of FTase is important in
designing of new potent enzyme inhibitors.
Pattern Recognition
•The FTase recognizes the CA1A2X at the C-terminal position of the RAS
protein:
CA1A2X
•C is the cysteine residue to which the prenyl group is attached;
•A1 and A2 are aliphatic amino acids;
•X is the carboxyl terminus specifying which prenyl group is attached
(geranylgeranyl or farnesyl group).
•The enzyme catalyzes also the transfer of the farnesyl group on the partial
tetrapeptide isolated from the main chain.
RAS Protein Posttranslational Modification
O
H
N
Val-Ile-Met-OH
O
FTase
SH
H
N
+
O
O
P
O
O
OP
Val-Ile-Met-OH
S
O-
O
1. Endoprotease
2. Methyltransferase
O
Palmitoylzation and
membrane localization
H
N
OMe
S
The Farnesyltransferase Crystals Structure
Water
molecules
a subunit
•The crystal structure of rat FTase
was resolved at 2.25 Å resolution.
• This protein is an heterodimer
consisting of 48 kD (a) and 46 kD
(b) subunits.
•The secondary structure of both a
and b subunits appears largely
composed of a-helices.
Zn
b subunit
•A single zinc ion, involved in
catalysis, is located at junction
between the hydrophilic surface of b
subunit and the hydrophobic deep
cleft of a subunit.
•The zinc is coordinated by three b
subunit residues and one water
molecule.
Classification of the FTase Ligands
FTase
Peptidomimetics
Substrates
Transition state
analogues
Activators
Inhibitors
FPP mimetics
Natural comp.
Catalytic
mechanism
Inhibition
mechanism
Pharmacophore
Computational Methods
•FTase crystal structure refinement
The structure was minimized using both
steepest descent algorithm until RMS = 0.5
and conjugated gradients until RMS = 0.01,
keeping backbone constrained to preserve
the experimental structure. The water
molecules are preserved in all simulations.
•Construction of the ligands
The conformational analysis was performed using high temperature (2000 K)
molecular dynamics (500 ps), which is able to span the conformational space
of flexible molecules. The best structure obtained was finally optimized by
MOPAC 6.0.
•Docking analysis
It was performed using BioDock: a software for automated docking of ligands
to biomacromolecules, based on a stochastic approach.
BioDock
The complex is bad
Ligand
Receptor
Random
rototranslation
of the ligand
NO
Complex
evaluation
New
complex
End of
docking
Cluster
analysis
YES
Cluster 1
Stop
Cluster 2
Cluster 3
Cluster n
CA1A2X Peptides
HS
H
N
+
H3N
O
N
H
O
H
N
HS
O
O-
+
H3N
S
O
Cys-Val-Ile-Met (CVIM)
O
H
N
N
H
O
O
H
N
O
O
OH
Cys-Val-Leu-Ser (CVLS)
NH
HS
H
N
H3N
O
O
N
H
H
N
O
HS
O
OS
Cys-Val-Trp-Met (CVWM)
H
N
H3N
O
O
N
H
H
N
O
O
OS
Cys-Val-Phe-Met (CVFM)
CVIM Peptide Conformations
CVIM - extended
dist. = 11.6 Å
CVIM - folded
dist. = 8.3 Å
CVIM Conformational Analysis
Activator
CVWM Conformational Analysis
Inhibitor
Conformational Analysis Results
From these results, we can suppose a hypothetical catalytic mechanism
consisting of two steps:
Recognition
Conformational
interconversion
Folded conformation
Activation
Extended conformation
Natural Inhibitors(1)
O
HO
O
COOH
O
O
O
OH
COOH
COOH
Zaragozic acid
H3C
IC50 = 12 nM
CH3
H3C
O
OH
MeO
O
O
H
H3C
H
H
OH
HO
CH2
H
CH3
O
O
H
O
CH3
O
O
O
O
Artemidolide
Fusidienol
IC50 = 360 nM
IC50 = 300 nM
Natural Inhibitors(2)
H3C
H3C
CH3
CH3
CH3
CH3
CH3
CH3
CH3
HOOC
CH3
HOOC
CH3
O
HOOC
O
Des-A
Des-B
IC50 = 0.9 mM
IC50 = 0.19 mM
CH3
CH3 O
CH3
R
O
HOOC
H3C
COOH
Z-Schizostatin
IC50 = 300 mM
H
O
COO-
CH3
OH
H
H3C CH3
Andrastatin A (R =CHO)
IC50 = 24.9 mM
Andrastatin B (R =CH2OH) IC50 = 47.1 mM
Andrastatin C (R =CH3)
IC50 = 13.3 mM
FTase - Fusidienol Complex
Alpha subunit
Beta subunit
Site Selectivity
Compound
Type
VO%CVLS
VO%FPP
CVLS
-
100
0
FPP
-
0
100
Fusidienol
N.S.
15,7
17,7
Zaragozic acid
N.S.
41,7
41,3
Andrastatin A
CVLS
43,3
6,1
Andrastatin B
CVLS
41
11,9
Andrastatin C
CVLS
44
6
Arteminolide
CVLS
47,3
26,9
Clav-A 1S,2R
CVLS
54,7
4,4
Clav-B 1S,2R
FPP
26,5
39.3
Schizostatin Z
FPP
10
36.6
Schizostatin E
FPP
7.1
27.8
Inhibition mechanism (Type): N.S. (not-selective), CVLS (peptidomimetic), FPP (FPPmimetic).
Classification of the Natural Inhibitors
Zaragozic Acid
Non specific pos.
Fusidienol
 volume
 VCVLS VFPP
Zn++ shielding
 VCVLS  VFPP
Natural inhibitors
Peptidomimetic
 VCVLS VFPP
Artemidolide
 lipole
FPP-mimetic
 VCVLS VFPP
Schizostatin
The Lipole
The lipole is calculated as sum of local values of logP, like dipolar
momentum:
L   ri   i
i
Where:
ri is the distance between atom i and the geometric center of the molecule;
li is the atomic value of the lipophilicity of atom i.
Lipole and Site Selectivity
Compound
Type
Lipole
LogP
CVLS
-
2.2
-0.5
FPP
-
-
-
Fusidienol
N.S.
1.4
1.8
Zaragozic acid
N.S.
0.8
2.0
Andrastatin A
CVLS
2.2
1.7
Andrastatin B
CVLS
2.2
1.6
Andrastatin C
CVLS
2.5
2.7
Arteminolide
CVLS
2.5
1.8
Clav-A 1S,2R
CVLS
2.1
6.0
Clav-B 1S,2R
FPP
4.3
4.2
Schizostatin Z
FPP
6.7
1.4
Schizostatin E
FPP
6.0
1.3
Lipole < 2.0
Non-specific inhibitors
2.0 < Lipole < 4.0
Peptidomimetics
Lipole > 4.0
FPP-mimetics
VEGA and the Lipole Calculation
VEGA Main Features
VISUALIZATION
File Conversion
Data
Interchange
Force field
attribution
Docking
analysis
Surface Mapping
Shape
Analysis
Web
Publishing
Property
Calculation
Trajectory Analysis
Dynamic
Animation
Time
Profiling
Flexibility
analysis
Pharmacophoric Model
Arg-202b
Arg-291b
N
H
NH2 +
OH
HN
NH2
H2N
Zn++
+ NH
2
Electron
rich zone
Tyr-300b
Tyr-361b
NH
HO
Aromatic
functions
HN
N
N
His-201a
+
NH3
His-248b
Amidic
groups
Lys-164a
+
NH3
Lys-356b
PHARMACOPHORIC
GROUPS
Acknowledgments
Bernard Testa
Luigi Villa
Anna Maria Villa
Lidia Perri
Eleonora Vocaturo
Antonio Boccardi
http://users.unimi.it/~ddl