The upcoming release of FieldStere will enable fragment growing, This presentation will introduce the current operation and results then focus on the new features that enable more complex bioisostere searching experiments.

1. Evaluating Bioisosteric Replacements Taking the Whole Molecule into Account Mark Mackey

2. Current Versions

3. Field Points Condensed representation of electrostatic, hydrophobic and shape properties (“protein’s view”) Molecular Field Extrema (“Field Points”) = Positive = Negative = Shape = Hydrophobic 3D Molecular Electrostatic Potential (MEP) Field Points 2D

4. +ve ionic H-bond acceptor Aromatic p cloud ‘H acceptor’ -ve ionic H-bond donor Hydrophobes Aromatic in-plane ‘H donor’ “Stickiest” surfaces (high vdW) Field points give you new insights into your molecule Explanatory Power of Fields = Positive = Negative = Shape = Hydrophobic Field point size show importance

5. XEDs make Fields Work Field patterns from Cresset’s proprietary XED force field reproduce experimental results Not using XEDs Interaction of Acetone and Any-OH from small molecule crystal structures Experimental Using XEDs XED adds p-orbitals to get better representation of atoms

6. Information Provided by Fields Structure Fields Experimental Field points give you new insights into your molecule

7. Comparing Molecules Clique based initial alignment Uses the distance matrix of Field Points Detailed score Single point Uses Field-Point on Field Scoring Optional simplex based optimisation of alignment Rigid body - no bonds twisted Uses detailed score as optimisation parameter

8. N-methyl acetamide Imidazole Field Alignment

9. N-methyl acetamide Imidazole Field Scoring To score a particular alignment, we use the field points of molecule 1 to sample the actual field of molecule 2 Cheeseright et al, J. Chem Inf. Mod., 2006, 665

10. Field Scoring N-methyl acetamide Imidazole To score a particular alignment, we use the field points of molecule 1 to sample the actual field of molecule 2 and vice-versa Cheeseright et al, J. Chem Inf. Mod., 2006, 665

11. Bioisosteres – PDE III Biologically relevant method for comparing molecules Bioisosteres Bioisosteric groups

12. FieldStere Finding bioisosteres by replacing sections of the molecule Valdecoxib Etoricoxib Rofecoxib 12nM

13. FieldStere’s Approach Select a region to replace and remove these atoms

14. FieldStere’s Approach Select a region to replace and remove these atoms Search database for matching fragments (geometric search only) (search runs on fragment conformations)

15. Select a region to replace and remove these atoms Search database for matching fragments (geometric search only) (search runs on fragment conformations) Form Products (minimise and add Field Points) FieldStere’s Approach

16. Select a region to replace and remove these atoms Search database for matching fragments (geometric search only) (search runs on fragment conformations) Form Products (minimise and add Field Points) Score FieldStere’s Approach 0.88

17. Produces more diverse, non-obvious bioisosteres Avoids fragment scoring limitations Allows for electronic influence of replacing a moiety on the rest of the molecule and vice versa Allows for neighbouring group effects Whole-Molecule Scoring Advantages

18. Where do you get fragments from? Fragment existing molecules Fragment at Heteroatoms C=O and C=S Bonds to rings Not NO2, COOH, etc Generate all sets of connected pieces Subject to MW, NH, and rotatable bond limits

19. Creating Fragments Conformation hunt done on all fragments

20. Database of Replacement Moieties Series of commercial compounds fragmented and recombined Resultant moieties placed into separate databases based on frequency V. Common Common Less Common Rare Very Rare Frequency roughly correlates with synthesizability Add your own database containing proprietary moieties

21. Example - COX-2 Search for Bioisosteres for cyclic lactone of Rofecoxib Search Common Dbs Actives: 9 of the first 10 clusters 21 of the first 30 clusters 87,225 frags

22. COX-2 Results

23. COX-2 Results

24. Product Space Result 1,484 Target Result 5

25. Cross Scoring Separate target and reference for scoring Bring in interactions from a parallel series Multiple series using different parts of a binding site Optimize a low activity series using a high activity “template” Grow molecules Mimic interactions of alternative series Fragment growing

26. Select a region to replace and remove these atoms Search database for matching fragments (geometric search only) (search runs on fragment conformations) Form Products (minimise and add Field Points) Score FieldStere’s Approach 0.68 0.78

27. Cross scoring example - BACE Multiple known actives for BACE Binding site has flexibility Ligand based methods less sensitive to protein movements Ligands interact with the catalytic Aspartates differently

28. PDB 2IQG - Hydroxyethylamine based inhibitor Complex chemistry Chiral PDB 3L59 - Small guanidine based inhibitor Excellent interactions with Asp’s BACE Experiment

29. 2IQG - Detail

30. 3L59 - Detail

31. Can we use 3L59 to improve 2IQG ? Use 2IQG as the “Target” Score replacements against 3L59 - the “Reference” Region to be replaced Only accept replacements with Carbon here

32. Preliminary Results Result 18 Result 31 Result 55 Result 83 Result 42 Result 110

34. Cross Scoring 2 - Fragment Growing FieldStere version 3.0.0 fragment growth example: P38 kinase bound to a fragment fluorescent probe PDB:3K3I specific to the ‘DFG-out’ conformation ‘DFG-in’ example with specificity towards the ‘Gly’ flipped hinge PDB:3ROC and/or 3HUB Selectivity potentially to be gained by combining ‘Gly flip’ and ‘DFG-out’ in one molecule Can we use the new version of FieldStere to grow the DFG-out fragment into the DFG-in hinge?* *we realize we don’t want to develop a fluorescent probe….but… as an illustration of utility it is OK. We also have a number of more reasonable edits of this fragment….

35. Fragment in DFG-out pocket, PDB:3K3I predominant hinge conformation

36. +Gly hinge flip ligand_1, PDB:3ROC Hinge Gly flip

37. +Gly hinge flip ligand_2, PDB:3HUB Hinge Gly flip

38. Fieldstere input Target was PDB: 3K3I Reference was PDB: 3ROC 20/80 scoring T to R All databases Accurate settings Size limit on fragment removed Fragments limited to 3 rotatable bonds Fragment must contain a ring

39. Fieldstere output: 2D mols

40. Fieldstere output: 3D mols Fragment and reference Rank 4 Rank 6 Rank 11 Rank 13 Rank 53

41. Fieldstere output: 3D mols and Fields Fragment and reference Rank 4 Rank 6 Rank 11 Rank 13 Rank 53

42. Fieldstere output: 3D overlay References and Rank 6

43. Outcome Fragment growth both possible and a facile process using FieldStere Interesting and sensible candidate molecules generated Many more options available for increasing diversity of the output

44. mark@cresset-bmd.com Questions welcomed