Molecular docking and QSAR analysis: a combined approach applied to FTase

1. 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

2. 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.

3. Pattern Recognition • The FTase recognizes the CA1A2X at the C-terminal position of the RAS protein: • 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.

4. RAS Protein Posttranslational Modification

5. 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 (a) and 46 kD (b) subunits. • The secondary structure of both a and b subunits appears largely composed of a-helices. • A single zinc ion, involved in catalysis, is located at junction between the hydrophilic surface of b subunit and thehydrophobic deep cleft of a subunit. • The zinc is coordinated by three b subunit residues and one water molecule. Water molecules a subunit Zn b subunit

6. FTase Peptidomimetics Substrates Activators Inhibitors FPP mimetics Transition state analogues Natural comp. Catalyticmechanism Inhibitionmechanism Pharmacophore Classification of the FTase Ligands

7. 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.

8. BioDock The complex is bad Ligand Randomrototranslation of the ligand Newcomplex Complexevaluation Receptor Clusteranalysis NO End ofdocking YES Cluster 1 Stop Cluster 2 Cluster 3 Cluster n

9. CA1A2X Peptides Cys-Val-Ile-Met (CVIM) Cys-Val-Leu-Ser (CVLS) Cys-Val-Phe-Met (CVFM) Cys-Val-Trp-Met (CVWM)

10. CVIM Peptide Conformations CVIM - extended dist. = 11.6 Å CVIM - folded dist. = 8.3 Å

11. CVIM Conformational Analysis Activator

12. CVWM Conformational Analysis Inhibitor

13. Conformational Analysis Results From these results, we can suppose a hypothetical catalytic mechanism consisting of two steps: Recognition Extended conformation Conformational interconversion Activation Folded conformation

14. Natural Inhibitors(1) Zaragozic acid IC50 = 12 nM Artemidolide IC50 = 360 nM Fusidienol IC50 = 300 nM

15. Natural Inhibitors(2) Des-A IC50 = 0.9 mM Des-B IC50 = 0.19 mM Z-Schizostatin IC50 = 300 mM Andrastatin A (R =CHO) IC50 = 24.9 mM Andrastatin B (R =CH2OH) IC50 = 47.1 mM Andrastatin C (R =CH3) IC50 = 13.3 mM

16. FTase - Fusidienol Complex Alpha subunit Beta subunit

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

18. Classification of the Natural Inhibitors Zaragozic Acid Fusidienol  volume Non specific pos.  VCVLSVFPP Zn++ shielding  VCVLS VFPP Natural inhibitors  lipole Peptidomimetic  VCVLSVFPP FPP-mimetic  VCVLSVFPP Artemidolide Schizostatin

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

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

21. VEGA and the Lipole Calculation

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

23. Pharmacophoric Model

24. Acknowledgments Bernard Testa Luigi Villa Anna Maria Villa Lidia Perri Eleonora Vocaturo Antonio Boccardi http://users.unimi.it/~ddl