Sorting Out HTS Hits by Protein Crystallography The case of the Macrophage Migration Inhibitory Factor Macrophage Migration Inhibitory Factor(MIF)
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
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
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 :
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 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)
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
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
MIF HTS Hits: Selected Compound Classes coumarins 1,3-benzoxazineso-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones
X-ray Analysis of MIF/HTS Hit Complexes P a= 67.9Å b= 68.0Å c= 88.5Å 1 MIF trimer / a.u. P 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 Å resolution (with CNX)
X-ray Structure of MIF Inactivated by CBR at 1.80Å resolution CBR SA-omit map Pro-1 CBR adduct MIF tautomerase active site
X-ray Structure of MIF Inactivated by CBR 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 7-hydroxycoumarin-3-carboxylic acid ethyl ester SA-omit map Tautomerase active siteOverall view
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
MIF HTS Hits: Selected Compound Classes coumarins 1,3-benzoxazines o-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones
X-ray Structure of MIF Inactivated by GP at 1.80Å resolution GP SA-omit map Tautomerase active site Pro-1 GP adduct
X-ray Structure of MIF Inactivated by GP 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
MIF HTS Hits: Selected Compound Classes coumarins 1,3-benzoxazines o-hydroxybenzylamines N-benzoylbarbituric acidsN-acylbenzothiazolones
X-ray Structure of MIF inactivated by GP or GP at 2.00/2.10Å resolution GP049459SA-omit maps GP Tautomerase active site Pro-1 GP adduct Pro-1 GP adduct
X-ray Structure of MIF inactivated by GP or GP 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 GP GP HCHO
Mass Spectrometry Analysis GP Obs MW=12,345Da M=0Da CBR Obs MW=12,449Da M=+104Da GP Obs MW=12,557Da M=+212Da GP Obs MW=12,557Da M=+212Da R Obs MW=12,457Da M=+112=+2x56Da CBR Obs MW=12,575Da M=+230Da GP 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
Enzymatic Studies rIC 50 : IC 50 relative to cis-p-coumaric acid Inhibition of MIF-catalysed tautomerisation of p-hydroxyphenylpyruvate at pH 6.5
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 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