1. Sorting Out HTS Hits by Protein Crystallography The case of the Macrophage Migration Inhibitory Factor Macrophage Migration Inhibitory Factor(MIF)

2. Organization of Drug Discovery Research  target identification  assay development  HTS  compound optimization  selection of drug candidate  structural genomics  assessment of « drugability »  screening by NMR  X-ray crystallographic screening  NMR analysis  X-ray analysis  SBDD cycle  SAR by NMR  hit validation  SAR analysis  lead selection

3. Sorting out the HTS hit list  Elimination of false positives:  hit confirmation (primary assay)  hit validation (secondary assay(s))  Structure validation  Classification into substance classes  Similarity searches  Generation of preliminary SAR data Lead selection

4.  X-ray analysis of representative HTS hit/protein target complexes  Validates a substance class, allows modelling of other class members  Reveals binding site, binding mode and mode of action  Reveals active ingredient (stereochemistry, etc …)  Guides lead optimization (SBDD)  Defines pharmacophore for database mining Sorting Out the HTS Hit List by Protein Crystallography :

5. The Case of the Macrophage Migration Inhibitory Factor (MIF)  pro-inflammatory cytokine involved in the immune response  anti-MIF antibodies  are protective in models of inflammatory diseases  block tumor progression and angiogenesis.  MIF knock-out animals are protected from high-dose LPS  MIF shows enzymatic (tautomerase) activity  Pro-1 is the catalytic residue

6. MIF 3D Structure  3 x 114 amino-acids  First X-ray structure solved in 1996 by Sun and Lolis (1MIF), Kato and Kuroki (1GIF) and Sugimoto and Nishihira (1FIM) MIF /p-hydroxyphenylpyruvate complex (2.5Å resolution; J.B. Lubetsky and E. Lolis; 1CA7.PDB)

7. Known Structural Homologs of MIF Human MIF (macrophage migration inhibitory factor) 3 x 114 aa Human DPT (dopachrome tautomerase) 3 x 117aa Pseudomonas p. CHMI (5-carboxymethyl- 2-hydroxymuconate isomerase) 3 x 125aa Pseudomonas p. 4-OT (4-oxalocrotonate tautomerase) 6 x 62 aa

8. Searching for MIF Tautomerase Inhibitors by HTS  > 320,000 compounds screened  49 hits validated  6 substance classes selected p-hydroxyphenylpyruvate Assay principle MIF keto form enol form

9. MIF HTS Hits: Selected Compound Classes coumarins 1,3-benzoxazineso-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones

10. X-ray Analysis of MIF/HTS Hit Complexes P2 1 2 1 2 1 a= 67.9Å b= 68.0Å c= 88.5Å 1 MIF trimer / a.u. P3 1 21 a= b= 96.1Å c= 105.0Å 1 MIF trimer / a.u.  Co-crystallization experiments performed with 20 HTS hits  8 structures solved (by molecular replacement)  Refined to 2.10Å - 1.50Å resolution (with CNX)

11. X-ray Structure of MIF Inactivated by CBR548621 at 1.80Å resolution CBR548621 SA-omit map Pro-1 CBR548621 adduct MIF tautomerase active site

12. X-ray Structure of MIF Inactivated by CBR548621 at 1.80Å resolution N-benzoylbarbituric acids are irreversible MIF tautomerase inhibitors that lead to benzoylation of the catalytic amino-terminal proline

13. X-ray Structure of the MIF/7-HCCEE Complex at 1.50Å resolution 7-hydroxycoumarin-3-carboxylic acid ethyl ester SA-omit map Tautomerase active siteOverall view

14. Analysis of the MIF/7-HCCEE Complex  design of a new scaffold ? Superposition with the p-hydroxyphenylpyruvate complex  Detailed analysis of the binding interactions  Identification of unexploited binding opportunities  Design of optimized derivative  Synthesis  In vitro assay  Biological assay  X-ray analysis SBDD

15. MIF HTS Hits: Selected Compound Classes coumarins 1,3-benzoxazines  o-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones

16. X-ray Structure of MIF Inactivated by GP049625 at 1.80Å resolution GP049625 SA-omit map Tautomerase active site Pro-1 GP049625 adduct

17. X-ray Structure of MIF Inactivated by GP049625 at 1.80Å resolution  the inactivation mechanism probably involves a quinone methide intermediate  o-hydroxybenzylamines are irreversible MIF tautomerase inhibitors that alkylate the catalytic amino-terminal proline

18. MIF HTS Hits: Selected Compound Classes coumarins  1,3-benzoxazines o-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones

19. X-ray Structure of MIF inactivated by GP049457 or GP049459 at 2.00/2.10Å resolution GP049459SA-omit maps GP049457 Tautomerase active site Pro-1 GP049459 adduct Pro-1 GP049457 adduct

20. X-ray Structure of MIF inactivated by GP049457 or GP049459 at 2.00/2.10Å resolution  1,3-benzoxazines, like o-hydroxybenzylamines, are irreversible MIF tautomerase inhibitors that alkylate the catalytic amino-terminal proline  1,3-benzoxazines decompose to o-hydroxybenzylamines prior to MIF alkylation GP049459 GP049457 - HCHO

21. Mass Spectrometry Analysis GP046972 Obs MW=12,345Da  M=0Da CBR548621 Obs MW=12,449Da  M=+104Da GP049457 Obs MW=12,557Da  M=+212Da GP049459 Obs MW=12,557Da  M=+212Da R244740 Obs MW=12,457Da  M=+112=+2x56Da CBR548224 Obs MW=12,575Da  M=+230Da GP049625 Obs MW=12,541Da  M=+196Da MDP14708 Obs MW=12,345Da  M=0Da  20 compounds analyzed in total  N-acylbenzothiazolones identified as irreversible MIF inhibitors

22. Enzymatic Studies rIC 50 : IC 50 relative to cis-p-coumaric acid Inhibition of MIF-catalysed tautomerisation of p-hydroxyphenylpyruvate at pH 6.5

23. Summary / Conclusions  the X-ray analysis of MIF/HTS hits co-crystals revealed an unexpected mode of action of several substance classes  the X-ray results prompted a careful evaluation of all HTS hits by mass spectrometry and enzymatic analysis  these studies allowed the identification of the most promising substance class  chemistry efforts could be redirected quickly Protein crystallography can greatly help sort out HTS hits !

24. Acknowledgements Novartis Biomedical Research Institute Basel, Switzerland Novartis Biomedical Research Institute Vienna, Austria Chemistry Philipp Lehr Peter Nussbaumer Erwin Schreiner Biology, Enzymology & Program Team Head Andreas Billich Protein Preparation Paul Ramage Mauro Zurini Mass Spectroscopy Francis Bitsch Rocco Falchetto Patrick Graff Crystallography Sylvie Raccuglia Joseph Rahuel