Fragment Screening in Drug Discovery

Comments

Transcription

Fragment Screening in Drug Discovery
Fragment Screening in Drug
Discovery
Marc Martinell
SEQT, Sitges, 19th-20th October 2006
Crystax Pharmaceuticals SL
Barcelona Science Park
Josep Samitier 1-5, E-08028 Barcelona
Tel: +34 93 403 4703 Fax : +34 93 403 4788 www.crystax.com
Overview
•
The Company
•
Fragment Screening
•
•
Fragment Library
•
Detection of Fragment Binding
•
Structures of fragment-protein complexes
•
Hit Selection and optimization
Summary
SEQT, 19-20th October 2006
1
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 leadfinding process.
•
Barcelona Science Park
Access to top technology equipment
and labs
X-ray Crystallography
NMR for Biomolecules
Fine Chemistry
Combinatorial Chemistry
Microcalorimetry
Surface Plasmon Resonance
Genomics and Transcriptomics
Proteomics
SEQT, 19-20th October 2006
Business Model
¾
¾
¾
Fee for Service
Co-Development of new drugs with pharma partners using our platform
technology
Development of own pipeline
Crystax’s approach to business
Fee-for-service
business and R&D
collaborations
• Deliver value to clients
• Develop relationships for
licensing opportunities
Product pipeline
from own drug
discovery
• Short term: in collaboration
with other companies
• Mid term: own licensing
opportunities
B2D2
Common technology platform
SEQT, 19-20th October 2006
2
Collaborations
SEQT, 19-20th October 2006
Fragment Screening
3
Fragment Screening
z
z
z
There is a massive amount of
drug like molecules with a
suitable molecular weight for
drug discovery
HTS 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.
1060 – 10180 Drug like molecules with Mw < 800
SEQT, 19-20th October 2006
Fragment Screening
z
z
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.
SEQT, 19-20th October 2006
4
Fragment Screening
Fragmentlike
µM
nM
pM
KD
Probability
mM
Drug-like
0
2
4
6
8
10
Ligand complexity
Probability of detection
Probability of binding
Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864
SEQT, 19-20th October 2006
Fragment Screening
Fragmentlike
µM
nM
pM
KD
Techniques able to
detect and develop low
affinity binders are
needed
Probability
mM
Drug-like
NMR
X-Ray Crystallography
Biophysical techniques
0
2
4
6
8
10
Ligand complexity
Probability of detection
Probability of binding
Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864
SEQT, 19-20th October 2006
5
Fragment Screening
SAR-by-NMR
(S.B. Shuker et al. Science 1996, 274, 1531-1534)
R. Carr and H. Jhoti, DDT, 2002, 7, 522-527
SEQT, 19-20th October 2006
Fragment Screening - Examples
Lead
Hit
N
O
p38 kinase
N
OH
IC50 = 1.1mM
N
O
N
OH
J. Fejzo et al, Chem.Biol, 1999,
6, 755-769
IC50 = 200nM
F
N
N
OH
Urokinase
NH
N
N
Ki = 56µM
NH2
V.L. Nienaber et al, Nat.
Biotech., 2000, 18, 1105-1108
Ki = 370nM
NH2
CO2H
Thymidylate
Synthase
O
O
O S
O S
N
N
CO2H
HN
CO2H
O
O
D. A. Erlanson et al, PNAS,
2000, 97, 9367-9372
Ki = 33nM
Ki = 1000µM
CO2H
SEQT, 19-20th October 2006
6
Fragment Library
Fragments are organic molecules
with a low degree of complexity
and non-reactive
CrystaX’s Fragment Library
commercial
compounds
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.
CrystaX’s
Fragment
Library
≈1000
3,000.000
SEQT, 19-20th October 2006
7
CrystaX’s Fragment Library
commercial
compounds
Molecular
properties
(fragment-like)
Unwanted
reactivity
Clustering
and
selection
CrystaX’s
Fragment
Library
≈1000
80.000
280.000
3,000.000
SEQT, 19-20th October 2006
CrystaX’s Fragment Library – Quality Control
Quality Control of individual compounds
• Solubility, identity, purity and stability
Quality Control of mixtures of 7-9 compounds
• Designed to obtain the minimum signal overlap among compounds
• Solubility, identity, purity and stability
615 compounds ready for Fragment Screening
in 71 mixtures
Constant monitoring of false
positive and/or promiscuous
binders
SEQT, 19-20th October 2006
8
Detection of fragment binding
Due to their low degree of
complexity, fragments are low
affinity binders
Fragment Screening by NMR
Target protein
• STD
• Relaxation edited spectra
NMR screening
• WaterLOGSY
• TrNOE
• 19F-NMR
• Chemical Shift Mapping (CSM)
Continuous
development
Positive Fragments
SEQT, 19-20th October 2006
9
Fragment Screening by NMR - STD
I0
Each molecule has characteristic
signals on a 1D 1H spectra
B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
SEQT, 19-20th October 2006
Fragment Screening by NMR - STD
I0
In a solution of a protein with a large
excess of these molecules, their
spectra is almost not affected
B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
SEQT, 19-20th October 2006
10
Fragment Screening by NMR - STD
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.
B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
SEQT, 19-20th October 2006
Fragment Screening by NMR - STD
I0
ISAT
By subtracting both spectra, an NMR
difference spectrum is obtained in
where ligand molecules that bind to
the target can be identified
ISTD = I0 - ISAT
δ (ppm)
B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
SEQT, 19-20th October 2006
11
Fragment Screening by NMR
The complete library is
screened by NMR
1D 1H mixture
Relaxation Edited Experiment
The signals of fragment
binders disappear
Saturation Transfer Difference (STD)
The signals of
fragment binders
appear
Direct deconvolution from
mixtures of fragments
SEQT, 19-20th October 2006
Fragment Screening by NMR – Examples
1D 1H spectrum
STD spectrum
SEQT, 19-20th October 2006
12
Fragment Screening by NMR – Examples
Direct Deconvolution
CXL-8
CXL-7
CXL-9
CXL-11
CXL-12
CXL-18
CXL-20
CXL-23
CXL-24
CXL-28
STD
SEQT, 19-20th October 2006
Fragment Screening by NMR – Examples
Direct Deconvolution
Positives fragments: CXL-20 and CXL-23
CXL-20
CXL-23
STD
SEQT, 19-20th October 2006
13
Fragment Screening by NMR – Examples
STD
STD upon
addition of known
active-site ligand
SEQT, 19-20th October 2006
Fragment Screening by NMR – Examples
CXL-23
CXL-23
Only compound
CXL-23 interacts
with the active site
STD
STD upon
addition of known
active-site ligand
SEQT, 19-20th October 2006
14
Fragment Screening by NMR – Examples
1D 1H mixture
Relaxation Edited Experiment
STD
The signals of fragment
binders disappear
The signals of fragment
binders appear
1D 1H compound CXL-212
Compound CXL-212
interacts with the
protein
SEQT, 19-20th October 2006
Fragment Screening by NMR – Examples
1D 1H
mixture
+ Competitor
Relaxation
Edited
Experiment
addition of known
active-site ligand
STD
1D 1H
compound
CXL-212
SEQT, 19-20th October 2006
15
Fragment Screening by NMR – Examples
1D 1H
mixture
Using competition studies by NMR
ligands for specific binding sites can be
identified
Relaxation
Edited
Experiment
STD
Compound CXL-212
interacts with the
active site of the
protein target
1D 1H
compound
CXL-212
SEQT, 19-20th October 2006
Projects at CrystaX
Project
Field
Hit rate
1
Inflammation
10%
2
Oncology
5%
3
Oncology
5%
4
Auto-immune
4%
5
Oncology
3%
6
-
1%
SEQT, 19-20th October 2006
16
Structures of fragment-protein
complexes
Due to their low affinity and small
size fragments are more difficult to
study by Xray crystallography
Fragment Screening by Xray
Positive Fragments
Crystallization (co-crystallization or soaking)
Structure determination
Fragment Hits
SEQT, 19-20th October 2006
17
High-Throughput Crystallography
Sparse matrix screening for
initial crystallization conditions
Reproduction of
known crystallization conditions
> 1000 conditions
Optimization of conditions
for crystal growth
Characterization
of crystals
Collection of
“apo”-datasets
Large scale production
of crystals
Ab-initio screening for
crystallization conditions
in the presence of inhibitors
Soaking of crystals
with ligands
Co-crystallization
under analogous conditions
Collection of diffraction data
in the presence of inhibitor
SEQT, 19-20th October 2006
Automatic Data Processing & Analysis of Results
z
z
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.
SEQT, 19-20th October 2006
18
Structures of fragment-protein complexes
Fragment Screening renders a high amount of structural data, thus increasing the
efficiency of the hit to lead process
SEQT, 19-20th October 2006
Projects at CrystaX
Project
Field
Hit rate
(NMR)
Hit
confirmation
(Xray)
1
Inflammation
10%
40%
2
Oncology
5%
45%
3
Oncology
5%
ongoing
SEQT, 19-20th October 2006
19
Alternative approaches
Primary screening method
Hit confirmation
NMR (ligand-based detection)
Xray
NMR (protein-based detection)
Xray
SPR
Xray
Biochemical assays
Xray
Virtual Screening
Xray
-
Xray
The combination of ligand-based NMR methods and Xray
crystallography renders the most general approach with the
minimum consumption of protein sample
SEQT, 19-20th October 2006
Hit Selection and
Optimization
Several prioritization criteria are
needed in order to select the most
interesting hits
20
Hit selection
Fragment Hits
Preliminar SAR
Hit validation and
selection
Selected HITS
Validation of Binding-mode
New chemical structures
Evaluation of other molecules within its
cluster and/or molecules that contain the
same binding motif
SEQT, 19-20th October 2006
Hit Optimization
Fragment Hits
Biophysical methods (B2D2TM)
• SPR Æ Kon and Koff
• Microcalorimetry Æ ∆Hº, ∆Sº
………
Selected HITS
Synthesis
Activity Assays
Optimization
LEADS
Molecular Modeling
Fragment Screening!!
SEQT, 19-20th October 2006
21
Biophysics-Based Drug Discovery (B2D2TM)
pM
Lead
Activity
Assays
nM
?
µM
Fragment
Screening
Hit
mM
SEQT, 19-20th October 2006
Biophysics-Based Drug Discovery (B2D2TM)
pM
Lead
Activity
Assays
nM
ADME
Toxicity
Selectivity
Patentability
µM
Fragment
Screening
B2D2TM
Hit
mM
Biophysical
characterization
renders high quality
data and increases
the efficiency of
Hit2Lead process
SEQT, 19-20th October 2006
22
Biophysics-Based Drug Discovery (B2D2TM)
Biophysics-Based Drug Discovery
B2D2TM
NMR
X-Ray crystallography
SEQT, 19-20th October 2006
Biophysics-Based Drug Discovery (B2D2TM)
Biophysics-Based Drug Discovery
B2D2TM
NMR
X-Ray crystallography
•
Calorimetry
•
•
•
∆G
∆H
-T∆S
∆G
∆H
Unique technique for
thermodynamic data (KD,
∆Hº, ∆Sº)
Low throughput
Label free
High protein consumption
-T∆S
Same KD but different
thermodynamics
SEQT, 19-20th October 2006
23
Use of ITC in Fragment Evolution
ITC requirements
10 < nKA[M]T < 100
Fragment Hits: mM - µM
KA ~ 104 – 108
KD ~ 100µM – 10nM
Soluble compounds
Lead compounds: nM - pM
High amounts of sample
SEQT, 19-20th October 2006
Use of ITC in Fragment Evolution
SEQT, 19-20th October 2006
24
Biophysics-Based Drug Discovery (B2D2TM)
Biophysics-Based Drug Discovery
B2D2TM
NMR
X-Ray crystallography
Calorimetry
Biacore (SPR)
RU
•
•
•
•
Kinetic data (KA, kon, koff)
Medium-high throughput
Immobilization needed
Low protein consumption
500
0
Same KD but
different kinetics
0
120
240
360
480
Time (seconds)
SEQT, 19-20th October 2006
Biophysics-Based Drug Discovery (B2D2TM)
Biophysics-Based Drug Discovery
B2D2TM
NMR
X-Ray crystallography
Calorimetry
Biacore (SPR)
Fluorescence spectroscopy
•
•
•
•
Affinity constant (KA)
Medium-high throughput
Fluorescent-label needed
Low protein consumption
SEQT, 19-20th October 2006
25
Summary
Renders….
Novel Structures
Fragment Screening
B2D2™
… but also….
SEQT, 19-20th October 2006
Summary
New binding modes
Novel Structures
New binding sites
Fragment Screening
B2D2™
Lead Optimization
Protein Hot-spots
Structural waters
Protein flexibility
Biophysical characterization
SEQT, 19-20th October 2006
26
Acknowledgements
Joan Aymamí
Miquel Coll
Maria Kontoyianni
Ingo Korndoerfer
Montse Soler
Xavier Barril
Isabel Navarro
Franck Chevalier
Teresa Luque
Irena 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 Moral
Marta Masip
Sarah Sotil
Verónica Toledo
Laura Quintana
Sonia Soriano
Anja Leimpek
Marian Domínguez
Marta Martín
Thank you for your
attention
27

Similar documents