1. Nov. 2006 B. S. Sadjad 1 Toward Fully Flexible Docking Bashir S. Sadjad bssadjad@uwaterloo.ca School of Computer Science, University of Waterloo, Canada Simulated Biomolecular Systems, Toronto, Canada

2. Nov. 2006 B. S. Sadjad 2 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

3. Nov. 2006 B. S. Sadjad 3 What Is Aspirin? ● Acetosal: ● Route of administration: Oral ● Structure: NOTE: Images without reference are taken from public domain (mostly Wikipedia)

4. Nov. 2006 B. S. Sadjad 4 How does Aspirin Work? ● A painkiller, also used against fever ● Reduce production of Prostaglandin, Thromboxane ● Prostaglandin: ● Prostaglandin binds to some trans-membrane proteins of spinal neuron cells causing pain.

5. Nov. 2006 B. S. Sadjad 5 How does Aspirin Work? (cont'd) ● Cyclooxygenase is inhibited (breaking the pathway). ● Thromboxane pathway: ● Many of drugs interfere in a protein function. ● Aspirin effect is irreversible, we like reversible!

6. Nov. 2006 B. S. Sadjad 6 Enzymes (Review) ● Enzymes are great catalyzers (may speed up a reaction by 5 to 17 order of magnitude). ● An explanation of how they work:

7. Nov. 2006 B. S. Sadjad 7 Enzyme Inhibition ● Change of shape or chemical properties of active site on an enzyme. ● Example: blocking active site in HIV protease by ritonavir.

8. Nov. 2006 B. S. Sadjad 8 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

9. Nov. 2006 B. S. Sadjad 9 How Was Aspirin Discovered? ● Roots can be traced back to 5 th century BC, in Hippocrates notes regarding a bitter powder extracted from willow bark easing pain. ● Similar references found in other ancient notes. Willow produces salicin which acts similarly to aspirin in the body. ● Main discovery was done in 19 th century.

10. Nov. 2006 B. S. Sadjad 10 Rational Drug Design ● The discovery was by trial and error anyway! ● How a similar drug may be discovered in 21 st century? – Identify related pathways – Select target proteins – Identify active sites and allosteric sites – Try to inhibit target proteins – Inhibition may happen by ligand binding

11. Nov. 2006 B. S. Sadjad 11 Rational Drug Design (Example) ● Zanamivir (a ligand) used for treatment of Influenza virus. ● Inhibits Neuraminidase, an enzyme on the surface of Influenza virus. ● One of the first rationally designed drugs, by Biota (1989). ● Marketed by GSK (1999). Neuraminidase crystal structure (the ligand is NOT Zanamivir)

12. Nov. 2006 B. S. Sadjad 12 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

13. Nov. 2006 B. S. Sadjad 13 High Throughput Screening (HTS) ● Once the target protein is purified a library of ligands might be tested against it. ● The size of a practical library is in 10 5 – 10 6 range. ● Ligands active against the target protein can be selected by automated mechanisms. ● This requires significant resources including expensive labs.

14. Nov. 2006 B. S. Sadjad 14 Virtual HTS ● How if we can predict the HTS result by a computer program? ● This is Virtual High Throughput Screening. ● One way to do this is by simulating the binding process using properties of involved molecules. ● Free Energy of Binding determines the affinity.

15. Nov. 2006 B. S. Sadjad 15 Free Energy ● Each conformation and binding mode has a specific free energy: image from [1]

16. Nov. 2006 B. S. Sadjad 16 Docking ● Determine the best binding mode: – An approximation of free energy is used (scoring function). – The search engine finds the minimum of the scoring function. ● Carbonic anhydrase and a bound ligand Image generated by CheVi of SimBioSys (coordinates from 1AZM PDB code)

17. Nov. 2006 B. S. Sadjad 17 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

18. Nov. 2006 B. S. Sadjad 18 Classification Criteria ● Type of scoring function: – Force-field based – Empirical – Statistical ● Search method: – Systematic – Stochastic ● Degree of ligand-protein flexibility

19. Nov. 2006 B. S. Sadjad 19 Scoring ● Approximating reality: a bad approximation won't work even with the best search method. ● A general form for an empirical scoring function: image from [2]

20. Nov. 2006 B. S. Sadjad 20 Search ● Stochastic and heuristic – MCDOCK (simulated annealing) – AutoDock2 (simulated annealing) – GOLD (genetic algorithm) ● Directed – FlexX (incremental construction) – eHiTS (exhaustive search) ● Combined – Glide (systematic pose gen. + stochastic optimization)

21. Nov. 2006 B. S. Sadjad 21 eHiTS ● eHiTS approach [4]: – Ligand fragmentation – Fragment rigid-dock – Fragment matching – Ligand reconstruction – Local optimization

22. Nov. 2006 B. S. Sadjad 22 Example (Rigid Docking) ● Each fragment is docked in cavity. ● For sufficient accuracy a fine sampling should be done.

23. Nov. 2006 B. S. Sadjad 23 Example (Matching) ● All fragments are scored. ● A diverse set of matching poses with high scores are selected. ● A full ligand pose is generated from each matching set.

24. Nov. 2006 B. S. Sadjad 24 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

25. Nov. 2006 B. S. Sadjad 25 Protein Structure and Ligand Binding ● Protein structure may significantly be changed by ligand binding. ● Calmodulin (a calcium-binding protein): – Movie: http://molmovdb.org/cgi-bin/morph.cgi?ID=78252- 5656 image from [1]

26. Nov. 2006 B. S. Sadjad 26 The Allosteric Effect ● Binding of ligands may regulate the protein function. ● Example: binding of oxygen and carbon-dioxide to hemoglobin:

27. Nov. 2006 B. S. Sadjad 27 Binding Site Flexibility ● Ligand binding changes the binding site of the protein. This is called induced fit. ● In many of protein-ligand complexes in PDB, the cavity surrounds the ligand with a small open part. ● Rigid treating of binding site (as done by most docking programs), makes binding energy prediction difficult.

28. Nov. 2006 B. S. Sadjad 28 Example (Ligand Binding) ● Conformational change at the binding site of Renin. image from [1]

29. Nov. 2006 B. S. Sadjad 29 Example (Cavity Closure) ● L-Arabinose- binding protein complexed with L-Arabinose. (PDB: 1ABE)

30. Nov. 2006 B. S. Sadjad 30 A Note on Structure-Function Assumption ● Amino Acid – Structure – Function assumption. ● Consider a highly hydrophilic protein sequence, is it folded in water? Does it have any functions? ● Indeed it is not in a single folded state but it can be functional! There are functional intrinsically unstructured proteins. ● They may fulfil different tasks and have different fold for each task.

31. Nov. 2006 B. S. Sadjad 31 Example (Unstructured) ● The pKID domain of CREB protein, complexed with KIX domain of CREB-binding protein. image from [3]

32. Nov. 2006 B. S. Sadjad 32 Example (Structural Change) ● The TAZ1 domain of CREB-binding protein complexed with two different domains. image from [3]

33. Nov. 2006 B. S. Sadjad 33 Outline ● How does Aspirin work?! ● Structure-based rational drug design ● What is Docking? ● Current approaches and software packages ● Why is protein flexibility important? ● One step forward in flexible docking...

34. Nov. 2006 B. S. Sadjad 34 Truly Flexible Docking ● A truly flexible docking application is in fact a folding program! ● eHiTS is an ab-initio method: folding complexity ● Different types of protein mobility: – Movement of large domains – Multiple conformations observed in a few residues

35. Nov. 2006 B. S. Sadjad 35 Movement of Domains ● Patterns of domain movement: ● Ribose-binding protein movie (2DRI, 1URP): – http://molmovdb.org/cgi-bin/morph.cgi?ID=645772-17065 image from [1]

36. Nov. 2006 B. S. Sadjad 36 Conformations of a Few Residues ● Acetylcholinesterase (PDB: 2ACE, 1EVE, 1VOT, 1ACL) image from [1]

37. Nov. 2006 B. S. Sadjad 37 Truly Flexible Docking ● A truly flexible docking application is in fact a folding program! ● eHiTS is an ab-initio method: folding complexity ● Different types of protein mobility: – Movement of large domains – Multiple conformations observed in a few residues (to be addressed in first step)

38. Nov. 2006 B. S. Sadjad 38 Binding Site Side-Chains ● Modeling side- chain flexibility of binding site residues in eHiTS. ● First the candidate chains should be selected. – Solvent exposed? – More statistics

39. Nov. 2006 B. S. Sadjad 39 Binding Site Side-Chains (cont'd) ● Same technique of fragmentation can be applied to side-chains. ● Rigid docking and pose matching with the backbone constraints.

40. Nov. 2006 B. S. Sadjad 40 The Problem Size (Difficulty) ● Run statistics for a set of 20 PDB codes (all numbers are averages): – # rigid fragments: 3.05 – # poses tried in RD: 60 million – # poses accepted in RD: 493,354 – Best Match RMSD: 0.60 A – Best Match Found: 1.01A (NOTE: Finding the best match is NP-hard for a general scoring function.)

41. Nov. 2006 B. S. Sadjad 41 Pose Match Example ● Closest match for an HIV protease inhibitor (1AAQ):

42. Nov. 2006 B. S. Sadjad 42 Training ● eHiTS uses a statistical scoring function. ● Training is done by known structures. ● Pose Match specific training is done by linear programming modeling and using CLP package. ● For receptor flexibility modeling we can either: – Generate receptor decoys – Use PDB complexes with same receptor

43. Nov. 2006 B. S. Sadjad 43 Goals and Previous Works ● Induced fit modeling in Glide [5]: – Docking into rigid receptor using softened scoring func. – Receptor active site sampling – Complex optimization (minor backbone flexibility) ● Differences with our approach: – Simultaneous handling of ligand/receptor flexibility – Same scoring function (no softened version) ● The set of cross-docking data can be used for training and benchmarking.

44. Nov. 2006 B. S. Sadjad 44 Selected References 1.S. J. Teague, Implications of Protein Flexibility for Drug Discovery, Nature Reviews (Drug Discovery), vol. 2, pp. 527-541, 2003. 2.D. B. Kitchen, H. Decornez, J. R. Furr, J. Bajorath, Docking and Scoring in Virtual Screening for Drug Discovery: Methods and Applications, Nature Reviews (Drug Discovery), vol. 3, pp. 935-949, 2004. 3.H. J. Dyson, P. E. Wright, Intrinsically Unstructured Proteins and Their Function, Nature Reviews (Molecular Cell Biology), vol. 6, pp. 197-208, 2005. 4.Z. Zsoldos, D. Reid, A. Simon, B. S. Sadjad, A. P. Johnson, eHiTS: A New Fast, Exhaustive Flexible Ligand Docking System, J. of Mol. Graphics and Modeling, (to appear – available online). 5.W. Sherman, T. Day, M. P. Jacobson, R. A. Friesner, R. Farid, Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects, J. Med. Chem., vol. 49, pp. 534-553, 2006.

45. Nov. 2006 B. S. Sadjad 45 Questions?