sorting out hts hits by protein crystallography the case of the macrophage migration inhibitory...
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Sorting Out HTS Hits by Protein CrystallographySorting Out HTS Hits by Protein Crystallography
The case of theThe case of the
Macrophage Migration Inhibitory Factor Macrophage Migration Inhibitory Factor
(MIF)(MIF)
Organization of Drug Discovery ResearchOrganization 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 listSorting 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 selectionLead selection
X-ray analysis of representative HTS hit/protein target complexesX-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 :Sorting Out the HTS Hit List by Protein Crystallography :
The Case of the Macrophage Migration Inhibitory Factor The Case of the Macrophage Migration Inhibitory Factor (MIF)(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 StructureMIF 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 complexMIF /p-hydroxyphenylpyruvate complex (2.5Å resolution; J.B. Lubetsky and E. Lolis; 1CA7.PDB)
Known Structural Homologs of MIFKnown Structural Homologs of MIF
Human MIF
(macrophage migrationinhibitory factor)
3 x 114 aa
Human DPT
(dopachrometautomerase)
3 x 117aa
Pseudomonas p. CHMI
(5-carboxymethyl-2-hydroxymuconate isomerase)
3 x 125aa
Pseudomonas p. 4-OT
(4-oxalocrotonatetautomerase)
6 x 62 aa
Searching for MIF Tautomerase Inhibitors by HTSSearching for MIF Tautomerase Inhibitors by HTS
> 320,000 compounds screened
49 hits validated
6 substance classes selected
OH
H
OH
OH
O
OHO
OH
O
p-hydroxyphenylpyruvate
Assay principleAssay principle
MIF keto formenol form
MIF HTS Hits: Selected Compound ClassesMIF HTS Hits: Selected Compound Classes
coumarins
1,3-benzoxazines o-hydroxybenzylamines
N-benzoylbarbituric acids N-acylbenzothiazolones
OOH O
NN
OO
O
OH
S
N
O
O
O
N
OR'
R
OH
NH
OR'
R
X-ray Analysis of MIF/HTS Hit ComplexesX-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)
X-ray Structure of MIF Inactivated by CBR548621X-ray Structure of MIF Inactivated by CBR548621at 1.80Å resolutionat 1.80Å resolution
NN
OO
O
OH
CBR548621 SA-omit map
Pro-1
CBR548621adduct
MIF tautomerase active site
X-ray Structure of MIF Inactivated by CBR548621X-ray Structure of MIF Inactivated by CBR548621at 1.80Å resolutionat 1.80Å resolution
N-benzoylbarbituric acids are irreversible MIF tautomerase inhibitors that lead to benzoylation of the catalytic amino-terminal proline
NO
O
(MIF)
O N N
OO
O
+ MIF
Pro-1
X-ray Structure of the MIF/7-HCCEE ComplexX-ray Structure of the MIF/7-HCCEE Complexat 1.50Å resolutionat 1.50Å resolution
O
O
O
OH O
7-hydroxycoumarin-3-carboxylicacid ethyl ester
SA-omit map
Tautomerase active siteOverall view
Analysis of the MIF/7-HCCEE Complex Analysis of the MIF/7-HCCEE Complex
design of a new scaffold ?
Superposition with thep-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 ClassesMIF HTS Hits: Selected Compound Classes
coumarins
1,3-benzoxazines o-hydroxybenzylamines
N-benzoylbarbituric acids N-acylbenzothiazolones
OOH O
NN
OO
O
OH
S
N
O
O
O
N
OR'
R
OH
NH
OR'
R
X-ray Structure of MIF Inactivated by GP049625X-ray Structure of MIF Inactivated by GP049625at 1.80Å resolutionat 1.80Å resolution
NH
OH
O
N
GP049625 SA-omit map
Tautomerase active site
Pro-1
GP049625adduct
X-ray Structure of MIF Inactivated by GP049625X-ray Structure of MIF Inactivated by GP049625at 1.80Å resolutionat 1.80Å resolution
the inactivation mechanism probably involves a quinone methide intermediate
N
OH
O
(MIF)
NH
OH
O
N
+ MIF
Pro-1
NH
R
OH
NH2 R
O
NH
R
N
OH
R
+
o-hydroxybenzylamines are irreversible MIF tautomerase inhibitors that alkylate the catalytic amino-terminal proline
MIF HTS Hits: Selected Compound ClassesMIF HTS Hits: Selected Compound Classes
coumarins
1,3-benzoxazines o-hydroxybenzylamines
N-benzoylbarbituric acids N-acylbenzothiazolones
OOH O
NN
OO
O
OH
S
N
O
O
O
N
OR'
R
OH
NH
OR'
R
X-ray Structure of MIF inactivated by GP049457 or GP049459 X-ray Structure of MIF inactivated by GP049457 or GP049459 at 2.00/2.10Å resolutionat 2.00/2.10Å resolution
GP049459 SA-omit maps
GP049457
N
O
O
O
N
NH
OH
O
O
N
Tautomerase active site
Pro-1
GP049459adduct
Pro-1
GP049457adduct
X-ray Structure of MIF inactivated by GP049457 or GP049459 X-ray Structure of MIF inactivated by GP049457 or GP049459 at 2.00/2.10Å resolutionat 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
N
OH
O
O
(MIF)
O
N
NH
OH
O
+ MIF
Pro-1
N
OH
O
O
(MIF)N
O
O
O
N
+ MIF
Pro-1
GP049459
GP049457
- HCHO
Mass Spectrometry AnalysisMass Spectrometry Analysis
O OOH
GP046972Obs MW=12,345DaM=0Da
N
O
N
O
O
O
CBR548621Obs MW=12,449DaM=+104Da
NH
OH
O
O
N
GP049457Obs MW=12,557DaM=+212Da
N
O
O
O
N
GP049459Obs MW=12,557DaM=+212Da
N
S O
O
R244740Obs MW=12,457DaM=+112=+2x56Da
N
O
NH
O
OO
I
CBR548224Obs MW=12,575DaM=+230Da
NH
OH
O
N
GP049625Obs MW=12,541DaM=+196Da
O O
O
O
O
OH
MDP14708Obs MW=12,345DaM=0Da
20 compounds analyzed in total N-acylbenzothiazolones identified as irreversible MIF inhibitors
Enzymatic StudiesEnzymatic Studies
Class Compound rIC 50 K i
[nM]Mode of inhibition
coumarins GP046972 0.10 450 Reversible, Competitive
MDP14708 0.086 320 Reversible, Competitive
o-hydroxybenzylamines GP049625 0.019 - Irreversible
GP49457 0.025 - Irreversible
1,3-benzoxazines GP049459 0.031 - Irreversible
N-benzoylbarbituric acids CBR548224 0.15 - Irreversible
CBR548621 0.04 - Irreversible
N-acylbenzothiazolones R244740 0.46 - Irreversible
rIC50: IC50 relative to cis-p-coumaric acid
Inhibition of MIF-catalysed tautomerisation of p-hydroxyphenylpyruvate at pH 6.5
Summary / ConclusionsSummary / 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 !
AcknowledgementsAcknowledgements
Novartis BiomedicalResearch InstituteBasel, Switzerland
Novartis BiomedicalResearch InstituteVienna, Austria
Chemistry
Philipp LehrPeter NussbaumerErwin Schreiner
Biology, Enzymology& Program Team Head
Andreas Billich
Protein Preparation
Paul RamageMauro Zurini
Mass Spectroscopy
Francis BitschRocco FalchettoPatrick Graff
Crystallography
Sylvie RaccugliaJoseph Rahuel