Sorting Out HTS Hits by Protein Crystallography

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

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

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. Sorting Out the HTS Hit List by Protein Crystallography : • 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

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 MIF /p-hydroxyphenylpyruvate complex (2.5Å resolution; J.B. Lubetsky and E. Lolis; 1CA7.PDB) • 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)

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 Assay principle enol form MIF keto form p-hydroxyphenylpyruvate • > 320,000 compounds screened • 49 hits validated • 6 substance classes selected

9. MIF HTS Hits: Selected Compound Classes coumarins N-benzoylbarbituric acids N-acylbenzothiazolones 1,3-benzoxazines o-hydroxybenzylamines

10. X-ray Analysis of MIF/HTS Hit Complexes P212121 a= 67.9Å b= 68.0Å c= 88.5Å 1 MIF trimer / a.u. P3121 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 CBR548621at 1.80Å resolution CBR548621 adduct Pro-1 SA-omit map CBR548621 MIF tautomerase active site

12. X-ray Structure of MIF Inactivated by CBR548621at 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 Complexat 1.50Å resolution SA-omit map 7-hydroxycoumarin-3-carboxylic acid ethyl ester Overall view Tautomerase active site

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

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

16. X-ray Structure of MIF Inactivated by GP049625at 1.80Å resolution GP049625 adduct Pro-1 SA-omit map GP049625 Tautomerase active site

17. X-ray Structure of MIF Inactivated by GP049625at 1.80Å resolution • o-hydroxybenzylamines are irreversible MIF tautomerase inhibitors that alkylate the catalytic amino-terminal proline • the inactivation mechanism probably involves a quinone methide intermediate

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

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

20. X-ray Structure of MIF inactivated by GP049457 or GP049459 at 2.00/2.10Å resolution GP049459 - HCHO GP049457 • 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

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

22. Enzymatic Studies Inhibition of MIF-catalysed tautomerisation of p-hydroxyphenylpyruvate at pH 6.5 rIC50: IC50 relative to cis-p-coumaric acid

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