Molecular docking and QSAR analysis:

a combined approach applied to FTase

inhibitors and 1a-AR antagonists

Università degli Studi di Milano

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 anti-cancer 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

NH

SH

O

Val-Ile-Met-OH

1. Endoprotease2. Methyltransferase

FTase

NH

S

O

OMe

+

OP

OP

O

O O

O O- -

-

Palmitoylzation andmembrane localization

NH

S

O

Val-Ile-Met-OH

The Farnesyltransferase Crystals Structure

•The crystal structure of rat FTase was resolved at 2.25 Å resolution.

• This protein is an heterodimer consisting of 48 kD () and 46 kD () subunits.

•The secondary structure of both and subunits appears largely composed of -helices.

•A single zinc ion, involved in catalysis, is located at junction between the hydrophilic surface of subunit and thehydrophobic deep cleft of subunit.

•The zinc is coordinated by three subunit residues and one water molecule.

subunit

subunit

Zn

Watermolecules

Classification of the FTase Ligands

FTaseFTase

PeptidomimeticsPeptidomimetics

FPP mimeticsFPP mimetics

Natural comp.Natural comp.

InhibitorsInhibitorsActivatorsActivatorsSubstratesSubstrates

Transition state analogues

Transition state analogues

Catalyticmechanism

Catalyticmechanism

Inhibitionmechanism

Inhibitionmechanism

PharmacophorePharmacophore

Computational Methods

•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.

•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.

BioDock

Randomrototranslation of the ligand

Randomrototranslation of the ligand

Complexevaluation

The complex is bad

Newcomplex

Newcomplex

LigandLigand

ReceptorReceptor

Clusteranalysis

Clusteranalysis

End ofdocking

NO

Cluster 1Cluster 1

Cluster 2Cluster 2

Cluster 3Cluster 3

Cluster nCluster n

YES

StopStop

CA1A2X Peptides

H3NNH

NH

NH

O

O

O

O

OSH

S

-+

Cys-Val-Ile-Met (CVIM)

H3N NH

SH

O

O

NH

O

NH

OH

O

O

+

Cys-Val-Leu-Ser (CVLS)

H3NNH

NH

NH

O

O

O

O

OSH

S

NH

-

Cys-Val-Trp-Met (CVWM)

H3NNH

NH

NH

O

O

O

O

OSH

S

-

Cys-Val-Phe-Met (CVFM)

CVIM Peptide Conformations

CVIM - extendeddist. = 11.6 Å

CVIM - foldeddist. = 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:

Conformationalinterconversion

Conformationalinterconversion

RecognitionRecognition Extended conformation

ActivationActivationFolded conformation

Natural Inhibitors(1)

OO

O OHO

OH

MeO

OO

OO

OH

COOH

OH

COOH

COOH

O

FusidienolIC50 = 300 nM

Zaragozic acidIC50 = 12 nM

OO

CH3

CH3

H

HCH3

H

O

OO

CH2

CH3

H

H

CH3 CH3

O OH

ArtemidolideIC50 = 360 nM

Natural Inhibitors(2)

O

H

CH3

HOCH3

CH3

CH3

O

CH3

CH3

CH3

COO-OH

R

Andrastatin A (R =CHO) IC50 = 24.9 M

Andrastatin B (R =CH2OH) IC50 = 47.1 M

Andrastatin C (R =CH3) IC50 = 13.3 M

CH3

O CH3

CH3

CH3

CH3

CH3

HOOC

Des-AIC50 = 0.9 M

CH3

O

CH3

CH3

CH3

CH3

HOOC

HOOC

Des-BIC50 = 0.19 M

COOH

HOOC

Z-Schizostatin IC50 = 300 M

FTase - Fusidienol Complex

Beta subunit

Alpha 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

Natural inhibitorsNatural inhibitors

FPP-mimetic VCVLS VFPP

FPP-mimetic VCVLS VFPP

Peptidomimetic VCVLS VFPP

Peptidomimetic VCVLS VFPP

lipole

Zn++ shielding VCVLS VFPP

Zn++ shielding VCVLS VFPP

Non specific pos. VCVLS VFPP

Non specific pos. VCVLS VFPP

volume

Zaragozic Acid Fusidienol

Artemidolide Schizostatin

The Lipole

The lipole is calculated as sum of local values of logP, like dipolar momentum:

i

iirL

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 < 2.0Non-specific inhibitors

2.0 < Lipole < 4.0Peptidomimetics

Lipole > 4.0FPP-mimetics

VEGA and the Lipole Calculation

File Conversion

VEGA Main Features

VISUALIZATION

Surface Mapping Trajectory Analysis

DataInterchange

Dockinganalysis

Force fieldattribution

ShapeAnalysis

WebPublishing

PropertyCalculation

DynamicAnimation

TimeProfiling

Flexibilityanalysis

Pharmacophoric Model

O H

Tyr-361

OH

Tyr-300

NH NH 2

N H 2+

NHNH 2

N H 2+

A rg -291

A rg -202

N

NH

N

NH

H is-201 H is-248

NH 3 NH 3

++

Lys-164 Lys-356

Zn++

E lectronrich zone

Am id icgroups

Arom aticfunctions

P H A R M A C O P H O R ICG R O U P S

Acknowledgments

Bernard Testa

Luigi Villa

Anna Maria Villa

Lidia Perri

Eleonora Vocaturo

Antonio Boccardi

http://users.unimi.it/~ddl