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 CA 1 A 2 X at the C-terminal position of the RAS protein: C is the cysteine residue to which the prenyl group is attached; A 1 and A 2 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

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 the  hydrophobic deep cleft of  subunit. The zinc is coordinated by three  subunit residues and one water molecule.  subunit  subunit Zn Water molecules

Classification of the FTase Ligands FTase Peptidomimetics FPP mimetics Natural comp. Inhibitors Activators Substrates Transition state analogues Catalytic mechanism Inhibition mechanism Pharmacophore

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 Random rototranslation of the ligand Complex evaluation The complex is bad New complex Ligand Receptor Cluster analysis End of docking NO Cluster 1 Cluster 2 Cluster 3 Cluster n YES Stop

CA 1 A 2 X Peptides Cys-Val-Ile-Met (CVIM) Cys-Val-Leu-Ser (CVLS) Cys-Val-Trp-Met (CVWM) 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: Conformational interconversion Conformational interconversion Recognition Extended conformation Activation Folded conformation

Natural Inhibitors (1) Fusidienol IC 50 = 300 nM Zaragozic acid IC 50 = 12 nM Artemidolide IC 50 = 360 nM

Natural Inhibitors (2) Andrastatin A (R =CHO)IC 50 = 24.9  M Andrastatin B (R =CH 2 OH)IC 50 = 47.1  M Andrastatin C (R =CH 3 )IC 50 = 13.3  M Des-A IC 50 = 0.9  M Des-B IC 50 = 0.19  M Z-Schizostatin IC 50 = 300  M

FTase - Fusidienol Complex Beta subunit Alpha subunit

Site Selectivity Inhibition mechanism (Type): N.S. (not-selective), CVLS (peptidomimetic), FPP (FPPmimetic).

Classification of the Natural Inhibitors Natural inhibitors FPP-mimetic  V CVLS  V FPP FPP-mimetic  V CVLS  V FPP Peptidomimetic  V CVLS  V FPP Peptidomimetic  V CVLS  V FPP  lipole Zn ++ shielding  V CVLS  V FPP Zn ++ shielding  V CVLS  V FPP Non specific pos.  V CVLS  V FPP Non specific pos.  V CVLS  V FPP  volume Zaragozic AcidFusidienol ArtemidolideSchizostatin

The Lipole The lipole is calculated as sum of local values of logP, like dipolar momentum: Where: r i is the distance between atom i and the geometric center of the molecule; l i is the atomic value of the lipophilicity of atom i.

Lipole and Site Selectivity Lipole < 2.0 Non-specific inhibitors 2.0 < Lipole < 4.0 Peptidomimetics Lipole > 4.0 FPP-mimetics

VEGA and the Lipole Calculation

File Conversion VEGA Main Features VISUALIZATION Surface MappingTrajectory Analysis Data Interchange Docking analysis Force field attribution Shape Analysis Web Publishing Property Calculation Dynamic Animation Time Profiling Flexibility analysis

Pharmacophoric Model

Acknowledgments Bernard Testa Luigi Villa Anna Maria Villa Lidia Perri Eleonora Vocaturo Antonio Boccardi