Sorting Out HTS Hits by Protein Crystallography

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


coumarins



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


coumarins



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

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Sorting Out HTS Hits by Protein Crystallography