Fragment Screening in Drug Discoverydrug discovery zHTS and traditional discovery techniques often start with relatively large and complex molecules. zThe main disadvantage of traditional

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Crystax Pharmaceuticals SLBarcelona Science Park

Josep Samitier 1-5, E-08028 Barcelona Tel: +34 93 403 4703 Fax : +34 93 403 4788 www.crystax.com

Fragment Screening in Drug Discovery

SEQT, Sitges, 19th-20th October 2006

Marc Martinell

SEQT, 19-20th October 2006

• The Company

• Fragment Screening

• Fragment Library

• Detection of Fragment Binding

• Structures of fragment-protein complexes

• Hit Selection and optimization

• Summary

Overview

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The Company

• CrystaX is a Structure-based Drug Discovery company.

• Founded in 2002 by recognised scientists J. Aymamí and M. Coll.

• Current team of 22, among them 12 PhD with international experience, additional technical staff.

• Advisory agreements with experts in complementary areas (Computational Chemistry, Organic Synthesis, etc.)

• Our main strength is the combination of structural biology and chemistry into a team that can address any issue in the lead-finding process.

Barcelona Science ParkAccess to top technology equipment

and labs

X-ray CrystallographyNMR for Biomolecules

Fine ChemistryCombinatorial Chemistry

MicrocalorimetrySurface Plasmon Resonance

Genomics and TranscriptomicsProteomics


Business Model

Fee for ServiceCo-Development of new drugs with pharma partners using our platformtechnologyDevelopment of own pipeline

Crystax’s approach to business

B2D2Common technology platform

Fee-for-service business and R&D

collaborations

Product pipeline from own drug

discovery • Short term: in collaboration

with other companies• Mid term: own licensing

opportunities

• Deliver value to clients• Develop relationships for

licensing opportunities

3


Collaborations

Fragment Screening

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1060 – 10180 Drug like molecules with Mw < 800

Fragment Screening

There is a massive amount of drug like molecules with a suitable molecular weight for drug discoveryHTS and traditional discovery techniques often start with relatively large and complex molecules.The main disadvantage of traditional approaches is that finding one right molecule amongst such a vast number is quite difficult and, moreover, it is indeed hard to, once found, jump from one chemical “branch” to another.


Fragment Screening

Fragment screening allows to start with a smaller molecule and then add as much complexity as needed.

The difficulty is that you start at a pre-hit stage, where functional activity is difficult/impossible to measure.

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Fragment Screening

Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864

Drug-likeFragment-like

0 2 4 6 8 10

Ligand complexity

Prob

abili

ty

Probability of detection Probability of binding

KDmM µM nM pM


Techniques able to detect and develop low

affinity binders are needed

NMRX-Ray CrystallographyBiophysical techniques

Fragment Screening

Drug-likeFragment-like

0 2 4 6 8 10

Ligand complexity

Prob

abili

ty

Probability of detection Probability of binding

KDmM µM nM pM

Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864

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Fragment Screening

R. Carr and H. Jhoti, DDT, 2002, 7, 522-527

SAR-by-NMR (S.B. Shuker et al. Science 1996, 274, 1531-1534)


Fragment Screening - Examples

Hit

NO

OH

N

N

O

OH

N

FIC50 = 1.1mMIC50 = 200nM

N

OH

NH2

SN

OO

CO2H

Ki = 56µM

Ki = 1000µM

N

NH

NH2

NN

Ki = 370nM

SN

OO

HN

O

CO2H

CO2H

O

CO2H

Ki = 33nM

Lead

p38 kinase

Urokinase

ThymidylateSynthase

D. A. Erlanson et al, PNAS, 2000, 97, 9367-9372

V.L. Nienaber et al, Nat. Biotech., 2000, 18, 1105-1108

J. Fejzo et al, Chem.Biol, 1999, 6, 755-769

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Fragment Library

Fragments are organic moleculeswith a low degree of complexityand non-reactive


CrystaX’s Fragment Library

Selection process based on the newest criteria for fragment libraries.

The balance between chemical space exploration and efficiency of the hit to lead process is optimized.

commercial compounds

3,000.000

≈1000

CrystaX’sFragment

Library

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CrystaX’s Fragment Library

Molecular properties

(fragment-like)

Unwanted reactivity

commercial compounds

3,000.000

280.000

80.000

Clustering and

selection

≈1000

CrystaX’sFragment

Library


CrystaX’s Fragment Library – Quality Control

Constant monitoring of false positive and/or promiscuous

binders

615 compounds ready for Fragment Screening in 71 mixtures

• Solubility, identity, purity and stability

Quality Control of individual compounds

• Designed to obtain the minimum signal overlap among compounds• Solubility, identity, purity and stability

Quality Control of mixtures of 7-9 compounds

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Detection of fragment binding

Due to their low degree ofcomplexity, fragments are lowaffinity binders


Target protein

NMR screening

Positive Fragments

• STD• Relaxation edited spectra• WaterLOGSY• TrNOE• 19F-NMR• Chemical Shift Mapping (CSM)

Continuousdevelopment

Fragment Screening by NMR

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Fragment Screening by NMR - STD

Each molecule has characteristic signals on a 1D 1H spectra

I0

B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890



I0 In a solution of a protein with a large excess of these molecules, their spectra is almost not affected


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I0

ISAT

When the protein is saturated with a selective irradiation, this saturation is transferred to the binding molecules. This saturation produces an attenuation of its NMR signal.




By subtracting both spectra, an NMR difference spectrum is obtained in where ligand molecules that bind to the target can be identified

I0

ISAT

ISTD = I0 - ISAT

δ (ppm)



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Saturation Transfer Difference (STD)

Fragment Screening by NMR

1D 1H mixture

Relaxation Edited Experiment The signals of fragment binders disappear

The signals of fragment binders appear

Direct deconvolution from mixtures of fragments

The complete library is screened by NMR


1D 1H spectrum

STD spectrum

Fragment Screening by NMR – Examples

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Direct Deconvolution

STD

CXL-23

CXL-20

CXL-18

CXL-12

CXL-11

CXL-9CXL-7

CXL-8

CXL-28

CXL-24



Direct Deconvolution

Positives fragments: CXL-20 and CXL-23

STD

CXL-23

CXL-20


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STD

STD upon addition of known active-site ligand



CXL-23 CXL-23

STD

STD upon addition of known active-site ligand

Only compound CXL-23 interacts

with the active site


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1D 1H mixture

Relaxation Edited Experiment

STD

1D 1H compound CXL-212

The signals of fragment binders disappear

The signals of fragment binders appear

Compound CXL-212 interacts with the

protein



addition of known active-site ligand

+ Competitor

1D 1H mixture

Relaxation Edited

Experiment

STD



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Compound CXL-212 interacts with the active site of the

protein target

Using competition studies by NMR ligands for specific binding sites can be

identified

1D 1H mixture

Relaxation Edited

Experiment

STD




3%Oncology5

1%-6

5%Oncology3

4%Auto-immune4

2

1

Project

Oncology

Inflammation

Field

5%

10%

Hit rate

Projects at CrystaX

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Structures of fragment-protein complexes

Due to their low affinity and smallsize fragments are more difficult tostudy by Xray crystallography


Fragment Screening by Xray

Positive Fragments

Crystallization (co-crystallization or soaking)

Structure determination

Fragment Hits

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Sparse matrix screening forinitial crystallization conditions

Optimization of conditionsfor crystal growth

Characterizationof crystals

Collection of“apo”-datasets

Large scale productionof crystals

Soaking of crystalswith ligands

Collection of diffraction datain the presence of inhibitor

Co-crystallizationunder analogous conditions

Ab-initio screening forcrystallization conditions

in the presence of inhibitors

Reproduction ofknown crystallization conditions

High-Throughput Crystallography

> 1000 > 1000 conditionsconditions


Automatic Data Processing & Analysis of Results

Data are processed automatically using commercial software and a modular suite of proprietary scripts.

Resulting electron densities are inspected individually, analyzed and classified. Models of the protein-ligand complex are partially or completely refined, depending on the needs of the individual project.

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Structures of fragment-protein complexes

Fragment Screening renders a high amount of structural data, thus increasing the efficiency of the hit to lead process


ongoing5%Oncology3

45%5%Oncology2

40%

Hit confirmation

(Xray)

1

Project

Inflammation

Field

10%

Hit rate (NMR)

Projects at CrystaX

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NMR (ligand-based detection) Xray

NMR (protein-based detection) Xray

SPR Xray

Xray

Biochemical assays Xray

-

Alternative approaches

The combination of ligand-based NMR methods and Xraycrystallography renders the most general approach with the

minimum consumption of protein sample

Primary screening method Hit confirmation

Virtual Screening Xray

Hit Selection and Optimization

Several prioritization criteria are needed in order to select the most interesting hits

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Hit validation and selection

Evaluation of other molecules within its cluster and/or molecules that contain the same binding motif

Hit selection

Selected HITS

Fragment Hits Preliminar SAR

Validation of Binding-mode

New chemical structures


Biophysical methods (B2D2TM)• SPR Kon and Koff• Microcalorimetry ∆Hº, ∆Sº

………

Synthesis

Molecular Modeling

Fragment Screening!!

Activity Assays

LEADS

Optimization

Selected HITS

Hit Optimization

Fragment Hits

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mM

µM

nM

pM

Fragment Screening

Activity Assays

Lead

Hit

?

Biophysics-Based Drug Discovery (B2D2TM)


mM

µM

nM

pM

Fragment Screening

Activity Assays

ADMEToxicity

SelectivityPatentability

B2D2TM

Lead

HitBiophysical

characterization renders high quality data and increases

the efficiency of Hit2Lead process


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Biophysics-Based Drug DiscoveryB2D2TM

NMR X-Ray crystallography





• Unique technique for thermodynamic data (KD, ∆Hº, ∆Sº)

• Low throughput• Label free• High protein consumption

Calorimetry

∆G ∆H -T∆S ∆G ∆H -T∆S

Same KD but differentthermodynamics


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Use of ITC in Fragment Evolution

10 < nKA[M]T < 100

ITC requirements

KA ~ 104 – 108

KD ~ 100µM – 10nM

Soluble compounds

High amounts of sample

Fragment Hits: mM - µM

Lead compounds: nM - pM


Use of ITC in Fragment Evolution

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• Kinetic data (KA, kon, koff)• Medium-high throughput• Immobilization needed• Low protein consumption

Calorimetry

Biacore (SPR)

Time (seconds)

0

500

0 120 240 360 480

RU

Same KD butdifferent kinetics





Calorimetry

Biacore (SPR)

Fluorescence spectroscopy

• Affinity constant (KA)• Medium-high throughput• Fluorescent-label needed• Low protein consumption


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Fragment ScreeningB2D2™

Summary

Novel Structures

Renders….

… but also….


Summary

Novel Structures

Lead Optimization

Structural watersProtein Hot-spots

New binding modes

Protein flexibility

New binding sites

Fragment ScreeningB2D2™

Biophysical characterization

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Joan AymamíMiquel Coll

Maria KontoyianniIngo KorndoerferMontse SolerXavier BarrilIsabel NavarroFranck ChevalierTeresa LuqueIrena Bonin

Unitat RMN (SCT-UB)Unitat Químic Fina (SCT-UB)Unitat Citometria (SCT-UB)Unitat Química Combinatòria (PCB)Plataforma Raigs-X (PCB)A. Llebaria (RUBAM, IIQAB-CSIC)R. Gutierrez (IMIM-UPF)F. J. Luque (UB)

Carolina MoralMarta MasipSarah SotilVerónica ToledoLaura QuintanaSonia SorianoAnja LeimpekMarian DomínguezMarta Martín

Acknowledgements

Thank you for your attention

Documents

Fragment Screening in Drug Discoverydrug discovery zHTS and traditional discovery techniques often start with relatively large and complex molecules. zThe main disadvantage of traditional