Sorting Out HTS Hits by Protein Crystallography The

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

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

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

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

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

MIF 3 D Structure MIF /p-hydroxyphenylpyruvate complex (2. 5Å resolution; J. B. Lubetsky and E. Lolis; 1 CA 7. PDB) · 3 x 114 amino-acids · First X-ray structure solved in 1996 by Sun and Lolis (1 MIF), Kato and Kuroki (1 GIF) and Sugimoto and Nishihira (1 FIM)

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

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

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

X-ray Analysis of MIF/HTS Hit Complexes P 212121 P 3121 a= 67. 9Å b= 68. 0Å c= 88. 5Å 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)

X-ray Structure of MIF Inactivated by CBR 548621 at 1. 80Å resolution CBR 548621 adduct Pro-1 SA-omit map CBR 548621 MIF tautomerase active site

X-ray Structure of MIF Inactivated by CBR 548621 at 1. 80Å resolution N-benzoylbarbituric acids are irreversible MIF tautomerase inhibitors that lead to benzoylation of the catalytic amino-terminal proline

X-ray Structure of the MIF/7 -HCCEE Complex at 1. 50Å resolution SA-omit map 7 -hydroxycoumarin-3 -carboxylic acid ethyl ester Overall view Tautomerase active site

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

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

X-ray Structure of MIF Inactivated by GP 049625 at 1. 80Å resolution GP 049625 adduct Pro-1 SA-omit map GP 049625 Tautomerase active site

X-ray Structure of MIF Inactivated by GP 049625 at 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

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

X-ray Structure of MIF inactivated by GP 049457 or GP 049459 at 2. 00/2. 10Å resolution GP 049459 adduct Pro-1 GP 049459 SA-omit maps GP 049457 adduct Tautomerase active site GP 049457 Pro-1

X-ray Structure of MIF inactivated by GP 049457 or GP 049459 at 2. 00/2. 10Å resolution GP 049459 - HCHO GP 049457 · 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

Mass Spectrometry Analysis GP 046972 Obs MW=12, 345 Da M=0 Da MDP 14708 Obs MW=12, 345 Da M=0 Da R 244740 Obs MW=12, 457 Da M=+112=+2 x 56 Da CBR 548621 Obs MW=12, 449 Da M=+104 Da GP 049625 Obs MW=12, 541 Da M=+196 Da CBR 548224 Obs MW=12, 575 Da M=+230 Da · 20 compounds analyzed in total · N-acylbenzothiazolones identified as irreversible MIF inhibitors GP 049457 Obs MW=12, 557 Da M=+212 Da GP 049459 Obs MW=12, 557 Da M=+212 Da

Enzymatic Studies Inhibition of MIF-catalysed tautomerisation of p-hydroxyphenylpyruvate at p. H 6. 5 r. IC 50: IC 50 relative to cis-p-coumaric acid

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 !

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