Transcript Fragment- based approaches to the design of candidate drugs that interrupt

Fragment- based approaches to the design of
candidate drugs that interrupt
protein-protein interactions
involved in cell regulation
O
H114 S161
E115
K91Q173
R93
D171
OH
NH2
O
NH2
Cdk6
N
FGFR
HGF
BRCA2
Drug-like
fragments
Tom Blundell, Chris Abell, Ashok Venkitaraman, Alfonso Martinez Arias, Luca
Pellegrini, Marko Hyvonen, Carol Robinson
University of Cambridge
Many difficult targets are found amongst
multiprotein complexes of
cell signalling and regulation
Growth
factor
cell membrane
RTK
P
Grb2
RAS
SOS
MAPKKK
cytoplasm
MAPKK
MAPK
nucleus
nucleus
MAPK
P
P
SRF Elk-1
SRE
ImmediateFos, jun etc
early genes
Further
complication:
often weak
binary
complexes
lead to
stable
multiprotein
complexes.
Structural biology and Drug Discovery
Target Selection
Lead Discovery
Lead Development
Target
Identification
& Validation
Screening
Sequence-structure
homology recognition
Structural genomics
Hits-to-leads
Structure-based
Screening
Lead
optimisation
Traditional use of
X-ray and modelled
3D structures
Complex of
cyclin D-dependent kinase Cdk6 bound to the
cell cycle inhibitor p19INK4d.
Large, flat and
uninteresting:
difficult to bind small
molecules?
Brotherton, Dhanaraj, Blundell, Laue, Nature 395, 244-250, 1998
STRATEGIES FOR
LEAD DISCOVERY WITH TARGETS THAT ARE
MULTIPROTEIN COMPLEXES
1. Exploitation of hot spots in protein-protein interaction
surfaces.
2. Formation of large aggregates of hydrophobic or
amphipathic molecules – a specific mechanism of nonspecific inhibition!
3. Exploitation of allosteric effects.
2. Fragment –based appoaches
Multiprotein Complexes
Preformed globular structures
Discontinuous epitopes
+
Preformed but adaptive globular structures
+
One or more partners become
structured on assembly
+
Continuous epitopes
1. RAD51 is an essential enzyme in DNA repair.
2. The breast cancer susceptibility protein
BRCA2 controls RAD51 activity via a direct
protein-protein interaction.
3. Disruption of the BRCA2-RAD51 binding
interface blocks RAD51 activity.
Expression and purification of
RAD51 for lead identification.
Screening:
1. NMR (in vitro)
2. X-ray (in crystal)
a
c
b
d
Alternative Fragment strategies
Growing
Linking or
coupling
Thrombin fragment identification
Novel hits
Novel binding sites
S1 Neutral Fragment
S2-S4 Fragment
• S2/S4 fragment
potency 10mM
•Linked S1 and S2/S4
fragment: new hybrid
compound
potency 200nM
Thrombin inhibition
OH
S4-pocket
OEt
O
O
NH
O
NH
N
S2-pocket
O
O
N
O
NH
NH
Ximelagatran
Exanta (AZ)
Melagatran
NH
NH
H2N
S1-pocket
HO N
H
• To identify novel, non-peptidic inhibitors which lack
strongly basic functionality
Fragment linking: thrombin inhibition
OH
S4-pocket
O
NH
O
N
S2-pocket
O
NH
NH
H2N
S1-pocket
S1 Fragment hits
S4-pocket
S2-pocket
N N
N
IC50 = 330 mM
N
Cl
S1-pocket
S2-S4 Ligands
S4-pocket
IC50 = 300 mM
MeO
O
S2-pocket
S O
HN
HO
NH2
N N
N
N
Cl
S1-pocket
Fragment Linking
S4-pocket
IC50 = 300 mM
MeO
O
S2-pocket
S O
HN
IC50 = 3.4 nM
HO
NN N
N
NN
NN
IC50 = 330 mM
NH
ClCl
S1-pocket
theoretical values
Kd (additive)
100 nM
Kd (superadditive) 0.1 nM
Biophysical chemist
Screen fragments against protein targets
using some of the following techniques:
1 1
(i) X-ray crystallography
(ii) NMR spectroscopy (Water LOGSY)
(iii) Isothermal titration calorimetry
(iv) Surface plasmon resonance
(v) Non-covalent mass spectrometry
Have recently opened a new facility
for Biological Chemistry which includes
new ITC and SPR equipment.
2
cocktail of three
fragments 0.5 mM
3
+ enzyme 20 mM
NH2
N N
N
S
N
1
fragment 1 only +
enzyme
Dynamic combinatorial chemistry (DCC)
Reactive fragments
Fragment combinations
Fragment combination bound
to protein target
Detect product either by
(i) Stopping equilibration and identifying
major species
(ii)Direct observation by x-ray crystallography
M. S. Congreve et al., Angew. Chemie Int.
Ed., 2003, 42, 4479-82
Targeting the RAD51:BRCA2
interaction with small molecules
Essential Phe and Ala side-chains
PheHisThrAlaSerGly
F-H-T-A-S-G
Lead identification
1. Strategy:
Find fragments that bind the Phe or Ala
pockets; link them
Screen fragment library by mass spec,
SPR, etc
2. Factors:
• Leads may have a high MW and/or a low
affinity
• Kd of the BRC:RAD51 interaction ~1µM in
preliminary SPR studies
3. Initial aim:
Leads with Kd ~1-10nM & most ‘drug-like’
characteristics
Proof-of-concept & optimization
studies for potential leads
2. Inhibition of RAD51 focus formation after
DNA damage
3. In vitro sensitization of cell lines to DNA
damaging agents
Increasing potency
1. Inhibition of the BRC peptide-RAD51
interaction in ELISA or FP assays
Potential clinical profiles
Genetic & cell biological studies demonstrate that
disruption of the BRCA2-RAD51 interaction • Suppresses DNA double-strand break repair by
homologous recombination during G2
• Sensitizes cells to DNA cross-linking agents, PARP
inhibitors, and replication blockers
So, potential clinical profiles could include • Combination with radiation or radiomimetics to
overcome G2 resistance
• Combination with DNA cross-linking agents, PARP
poisons or topoisomerase inhibitors
Parallel approaches to lead identification
Conventional screens
Fragment-based
screens
Synthetic
libraries
[MRCT]
Natural product
libraries
[EU]
Lead ID, proof-of-concept studies
Lead optimization
Pre-clin development
Research Scientist Appointments
1. Medicinal Chemist.
2. Biophysical Chemist
3. Molecular biologist/biochemist
4. X-ray crystallography
5. Cell biologist for assays
Biophysical chemist
Screen fragments against protein targets
using some of the following techniques:
1 1
(i) X-ray crystallography
(ii) NMR spectroscopy (Water LOGSY)
(iii) Isothermal titration calorimetry
(iv) Surface plasmon resonance
(v) Non-covalent mass spectrometry
Have recently opened a new facility
for Biological Chemistry which includes
new ITC and SPR equipment.
2
cocktail of three
fragments 0.5 mM
3
+ enzyme 20 mM
NH2
N N
N
S
N
1
fragment 1 only +
enzyme
Synthetic organic chemist
Preparation of fragment library
(analysis of chemical structures, knowledge of reactivity, solubility etc)
Strategy to build up from initial fragment hits to lead compounds (going
from mM to nM)
Preparation of successive generations of compounds as go from hits to
leads. Compound synthesis, purification and characterisation (specific
skil set, cannot be done by e.g. biochemist or molecular biologist)
Development of fragment combining approaches (including dynamic
combinatorial chemistry and dynamic combinatorial crystallography)
We have a recent success at carrying out fragment based drug
discovery in the University resulting in nM inhibitor (thrombin project)
Thrombin inhibition
OH
S4-pocket
OEt
O
O
NH
O
NH
N
S2-pocket
O
O
N
O
NH
NH
Ximelagatran
Exanta (AZ)
Melagatran
NH
NH
H2N
S1-pocket
HO N
H
• To identify novel, non-peptidic inhibitors which lack
strongly basic functionality
Fragment linking: thrombin inhibition
OH
S4-pocket
O
NH
O
N
S2-pocket
O
NH
NH
H2N
S1-pocket
S1 Fragment hits
S4-pocket
S2-pocket
N N
N
IC50 = 330 mM
N
Cl
S1-pocket
S2-S4 Ligands
S4-pocket
IC50 = 300 mM
MeO
O
S2-pocket
S O
HN
HO
NH2
N N
N
N
Cl
S1-pocket
Fragment Linking
S4-pocket
IC50 = 300 mM
MeO
O
S2-pocket
S O
HN
IC50 = 3.4 nM
HO
NN N
N
NN
NN
IC50 = 330 mM
NH
ClCl
S1-pocket
theoretical values
Kd (additive)
100 nM
Kd (superadditive) 0.1 nM
Biophysical chemist
Screen fragments against protein targets
using some of the following techniques:
1 1
(i) X-ray crystallography
(ii) NMR spectroscopy (Water LOGSY)
(iii) Isothermal titration calorimetry
(iv) Surface plasmon resonance
(v) Non-covalent mass spectrometry
Have recently opened a new facility
for Biological Chemistry which includes
new ITC and SPR equipment.
2
cocktail of three
fragments 0.5 mM
3
+ enzyme 20 mM
NH2
N N
N
S
N
1
fragment 1 only +
enzyme
Notch is a single transmembrane receptor activated by ligands of the DSL family
Ligand interaction triggers a series if regulated proteolytic events wich release the
intracellular domain of Notch to interacy with nuclear proteins in particular CSL
-secretase (PS)
Notch plays a central role in most (all) cell fate decision during development and the
differentiation of mES and hES cells
Alterations in Notch activity lead to
Alagile syndrome
Spondylocostal dysostosis
CADASIL
and in many instances
Cancer
T cell acute lymphoblastic leukemia (T-ALL)
Grabher et al. Nature Reviews Cancer advance online publication;
published online 13 April 2006 | doi:10.1038/nrc1880
-secretase has other substrates
Cadherin
ErbB-4
ß-amyloid precursor protein (APP)
Corollary: other processes might be affected
-secretase inhibits Notch activation and
thus has secondary effects on Notch signalling
in various places e.g intestine and the immune system
Notch
Corollary: one could use -secretase inhibitors to curtail CRC. However at the moment this results
in all cell differentiating and an dissappearance of the epithelium through exhaustion of the niche
In transgenic models induction of lymphomas through activation of Notch has a very long latency
which is shortened by secondary events. The reverse is also truth e.g Ikaros-Notch connection
Corollary: targetting Notch activation might not have an effect on endogenous Notch before
being effective on the lymphoma.
T cell acute lymphoblastic leukemia (T-ALL)
Grabher et al. Nature Reviews Cancer advance online publication;
published online 13 April 2006 | doi:10.1038/nrc1880
Endocytosis
Traffick
Stability
Transcription
-secretase has other substrates
Cadherin
ErbB-4
ß-amyloid precursor protein (APP)
-secretase inhibits Notch activation and
thus has secondary effects on Notch signalling
in various places e.g intestine and the immune system
In transgenic models induction of lymphomas
through activation of Notch has a very long
latency which is shortened by secondary events.
The reverse is also truth e.g Ikaros-Notch connection
Specificity: Notch ANK only interact
with Notch effectors. This allows
selective interference with Notch activity.
Targetting Notch ANK will not affect
Notch activation but rather active Notch.
It will be possible to target specific processes
e.g. transcription, traffick, degradation. It
Will diminish interference with normal
Notch signalling.
Targetting Notch ANK will allow a
particularly effective way of treating
pathologies triggered by promiscuous
transcriptional activity of Notch