1. DE NOVO DESIGN OF A THYMIDYLATE KINASE INHIBITOR

2. Notes dTMP is one of the building blocks for DNA synthesis Enzyme inhibition inhibits DNA synthesis and cell division Enzyme inhibitors are potential anticancer agents Inhibitors can be modelled on the substrate or cofactor Thymidylate Synthase Enzyme Reaction

3. Notes Provides a 1-C unit for biosynthetic pathways Inhibitors can be based on the cofactor structure Difficult to gain selectivity between enzymes using the same cofactor 5,10-Methylenetetrahydrofolate Dihydrofolic acid CofactorCofactor Thymidylate Synthase

4. Notes The design of a novel inhibitor based on the structure of the binding site Crystallize target enzyme with known inhibitors Establish structure by X-ray crystallography Molecular modelling studies to carry out following Identify binding site and binding regions Design structure to fit the binding site Incorporate functional groups to make binding interactions Possibility of better selectivity between different targets De Novo Design

5. Notes 5-Fluorodeoxyuridylate binds to the substrate binding site CB 3717 binds to the cofactor binding site Enzyme inhibitors CB 37175-Fluorodeoxyuridylate De Novo Design

6. Notes Identifies binding interactions of pteridine ring Identifies available amino acids and bridging water molecule Binding interactions for CB 3717 De Novo Design

7. Notes Remove CB 3717 in silico Set up a grid in the empty binding site Place an aromatic CH probe at each grid point Measure hydrophobic interactions The binding pocket for the pteridine ring is hydrophobic Identify a hydrophobic scaffold to fit the pocket Scaffold must be smaller than binding pocket to allow introduction of functional groups Add functional groups to make additional binding interactions De Novo Design

8. Notes Hydrophobic naphthalene group forms van der Waals interactions with the binding site Room for additional binding groups De Novo Design

9. Notes Lactam introduced to allow additional hydrogen bond interactions with the binding site Naphthostyryl scaffold De Novo Design

10. Notes Amino substituent added to gain access to vacant region Placed at 5-position for synthetic feasibility Can vary N-alkyl groups without introducing an asymmetric centre De Novo Design

11. Notes The benzyl group mimics the benzene ring of the cofactor De Novo Design

12. Notes Phenylsulfonylpiperazine group is added for water solubility Positioned to protrude from binding site Makes contact with surrounding water No desolvation penalty De Novo Design

13. Notes De novo designed inhibitor is now synthesized and tested Active inhibitor Co-crystallized with enzyme Crystal structure determined De Novo Design

14. Shift Water shifted Binding Interactions IntendedActual Notes Inhibitor binds deeper into pocket than expected Ala-263 shifted due to steric clash Water molecule displaced to different position Badinteraction

15. Notes Molecular modelling studies of actual binding interactions Identifies 4 regions for modification Possible analogues are overlaid with lead compound to test whether they fit the binding site Synthesis of analogues Structure-based Drug Design

16. Region R 1 Substituent fits hydrophobic pocket Pocket becomes hydrophilic with depth Polar functional group at the end of the alkyl chain may be beneficial CH 2 CH 2 OH has greater binding affinity Methyl better than ethyl Structure-based Drug Design

17. Region R 2 Carbonyl oxygen replaced with amidine group Capable of binding to Ala-263 instead of repelling it More basic and protonated - allows ionic interaction and stronger hydrogen bonding interaction Increased inhibition Binding interactions identified from crystal structure Structure-based Drug Design

18. Binding interactions Structure-based Drug Design Ionic and stronger H- bonding interactions H-Bonding Notes Binding interactions as expected Ala-263 not displaced Bridging water molecule not displaced

19. Region R 3 Small hydrophobic pocket available in the region Methyl or chloro-substituent both beneficial for activity Structure-based Drug Design

20. Region R 4 Morpholine ring found to be beneficial for selectivity and pharmacokinetics Structure-based Drug Design

21. Notes Structures are synthesized containing combinations of the optimum groups at each position Amidine is the most important group for enhanced activity Inclusion of all the optimum groups is not beneficial Structure-based Drug Design

22. Notes Amidine, morpholine and methyl group are introduced No change at R 3 Potent inhibitor (500 x more active) Structure taken forward for clinical trials Structure-based Drug Design

23. Principles De novo design is useful in designing lead compounds for structure- based drug design Designed structure should be ‘loose fitting’ and flexible Allows possibility of different binding modes if binding does not take place as predicted Allows scope for further modification and drug optimization Compounds should be synthetically feasible Compounds should be in a stable conformation Desolvation penalties need to be considered De Novo Design