DE NOVO DESIGN OF A THYMIDYLATE KINASE INHIBITOR

1. DE NOVO DESIGN OF A THYMIDYLATE KINASE INHIBITOR

2. ThymidylateSynthase EnzymeReaction • 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

3. 5,10-Methylenetetrahydrofolate Dihydrofolic acid ThymidylateSynthase Cofactor • 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

4. De Novo Design • 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

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

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

7. De Novo Design • 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

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

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

10. De Novo Design • 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

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

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

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

14. Bad interaction Shift Binding Interactions Intended Actual Water shifted • Notes • Inhibitor binds deeper into pocket than expected • Ala-263 shifted due to steric clash • Water molecule displaced to different position

15. Structure-based Drug Design • 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

16. Structure-based Drug Design • Region R1 • Substituent fits hydrophobic pocket • Pocket becomes hydrophilic with depth • Polar functional group at the end of the alkyl chain may be beneficial • CH2CH2OH has greater binding affinity • Methyl better than ethyl

17. Structure-based Drug Design • Region R2 • 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

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

19. Structure-based Drug Design • Region R3 • Small hydrophobic pocket available in the region • Methyl or chloro-substituent both beneficial for activity

20. Structure-based Drug Design Region R4 Morpholine ring found to be beneficial for selectivity and pharmacokinetics

21. Structure-based Drug Design • 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

22. Structure-based Drug Design • Notes • Amidine, morpholine and methyl group are introduced • No change at R3 • Potent inhibitor (500 x more active) • Structure taken forward for clinical trials

23. De Novo Design • Principles • De novo design isuseful 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