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Alessio Bortoluzzi
Alessio Ciulli Group
University of Dundee
22nd April 2015
Targeting Protein-Protein Interactions (PPIs)
Ubiquitin Field Chromatin Field
• A bump-and-hole approach to engineer controlled selectivity of
BET bromodomain chemical probes.
Science 2014, 346 (6209), 638
• Targeting Low-Druggability Bromodomains: Fragment Based
Screening and Inhibitor Design Against the BAZ2B Bromodomain.
J. Med. Chem. 2013, 56 (24), 10183
• ………………
• Structure-Guided Design and Optimization of Small Molecules
Targeting the PPI between the E3 Ubiquitin Ligase VHL and the
HIF Alpha Subunit with In Vitro Nanomolar Affinities.
J. Med Chem. 2014, 57 (20), 8657.
• Biophysical studies on interactions and assembly of full-size E3
ubiquitin ligase: SOCS2:ElonginBC:Cullin5:Rbx2.
J.Biol.Chem.2015, 209(7), 4178.
• ………………
Why Target Protein-Protein Interactions?
Most of human proteins do not work on their own but in complex with other
protein(s), PPIs are central to all the biological processes and often dysregulated
in disease – the possibility to modulate PPIs holds enormous therapeutic
potential.
Nat. Rev. Drug Discov. 2002 1(9), 727
Targeting Protein-Protein Interactions by Fragment-Based Drug Discovery (FBDD)
PPI interfaces are generally flat and large (~1,000-2,000 A2) compared to the deep pockets that typically
bind small molecules (~300-500 A2) and unlike enzymes or GPCRs do not offer starting small molecules
for the drug discovery process. (Chem. Biol. 2014 21(9), 1102)
PPIs are notoriously challenging targets and traditional approaches such High Throughput Screening
(HTS) often fail.
HTS
FBDDFragments are low-molecular-
weight molecules (~<300 Da)
with low chemical complexity.(Nat. Protoc. 2013 8(11), 2309)
Adapted from: Top. Curr. Chem. 2012 317, 145
Targeting Protein-Protein Interactions by Fragment-Based Drug Discovery (FBDD)
Nat. Protoc. 2013 8(11), 2309
Selected clinical-stage compounds originating
from fragment-based lead discovery
Nat. Rev. Drug Discov. 2013 12(1), 5
Advantages and limitations of FBDD and HTS
Adapted from: Top. Curr. Chem. 2012 317, 145
How to Screen for Fragments?
Fragment screening methods – Pool result:
Erlanson, D. (Ed.) Practical Fragments 2011
Why not BLI?
• Label free technique
• Provides information on
the binding kinetics
• Versatile
• Good throughput
• ……
• Low MW limit of
detection (~150 Da)
• Run a screen on a 140-fragments library in triplicate on
eIF4E: 8/9 hits found in all 3
repetitions.
JNK-1 28/34: hits found in all 3
repetitions.
• eIF4E screened against larger library – 6500 fragments
– and of the selected hits 50% were reconfirmed in a
second screen.
• eIF4E was screened in a biochemical assay and a 52%
hit overlap was observed with the hits from BLI.
Conclusion: BLI is suitable for
small molecules characterization
and fragment screening
Bio-Layer Interferometry (BLI) Technology for Fragment Screening
Fragment Screening Approach
PPI Target Fragment library
Differential Scanning
Fluorimetry (DSF)
Bio-Layer
Interferometry (BLI)
1D Ligand Observed NMR
(CPMG, WaterLOGSY and STD)
1° Step
Minimize the number
of false negative hits
2° Step
Remove false positive
hits
Overview
- Description of the techniques used in the fragment screening cascade (DSF, BLI
and NMR).
- Analysis of the outcome of three fragment screenings.
- Conclusions from our experience in using BLI as fragment screening tool.
Differential Scanning Fluorimetry (DSF) Screen
Binding of a compound to a protein will result in an increase in the melting temperature (Tm).
Fluorescent dye – typically SYPRO
Orange – that is quenched in
aqueous solution but highly
fluorescent in no-polar environment
(e.g. hydrophobic patches of
unfolded proteins)
Adapted from: Nat. Protoc. 2007 2(9), 2212
Target protein
Typical Tm shift induced by fragments is of 0.5-2 °C (Nat. Protoc. 2013 8(11), 2309)
A fragment is considered a hit if induces a ΔTm > 2 x s.d. sample control.
Bio-Layer Interferometry (BLI) Screen at Single Point Concentration
Target protein is biotinylated in a ~1:1 molar ratio using commercially available kits.
Target
Protein
B= Linker
B = Biotin
Super Streptavidin
(SSA) Biosensor
Loading
10-50 μg/mL
Sensor Tray Sample Plate (384 wells)
16 SSA Biosensor loaded
with B-Protein and
quenched with Biocytin
16 Reference SSA Biosensors
– blocked with 1 μg/mL of
Biocytin
200 = Fragment at 200 μM
B = Buffer
= Positive control – if available
• 154 fragments per plate
• 90 minutes per plate
• 1200 fragments screened in 12 hours
Reference
wells
Bio-Layer Interferometry (BLI) Screen at Single Point Concentration
Instrument: Octet RED 384
Typical cycles are: Baseline – 60 seconds; Association – 60 seconds and Dissociation – 60 seconds
Experiment performed at 25 °CRaw Data
Reference
Sensors
Protein-Loaded
Sensors
Data are processed by double reference subtraction to remove drift and well-to-well artifacts:
Threshold: median
plus 3 X robust s.d.
Hit rate is target
dependent – typically
20-100 fragments (out
of ~1200) are selected
for follow up
Bio-Layer Interferometry (BLI) Screen – Hit Confirmation
Selected fragments are rescreened at 6 points concentration – 3 fold dilution from 500 μM.
Sensor Tray Sample plate (384 wells)
Reference
wells
Raw Data
• 28 fragment per plate
• Typical cycles are:
Baseline – 60 seconds;
Association – 60 seconds;
Dissociation – 90 seconds
• 3 hours and 15 minutes per
plate.
Bio-Layer Interferometry (BLI) Screen – Hit Confirmation
Data are processed by double reference subtraction to remove drift and well-to-well
artifacts and visually inspected to select fragments that give a concentration dependent
response and to discard fragments with not ideal behavior :
500 μM
167 μM
55 μM
18 μM
6&2 μM
Concentration dependent response
Association step reach saturation
“Reasonable” dissociation rate
✕
✕
✓
~ 50% of the fragments selected during the single
point concentration screening are discarded
Hit Validation by 1D-Ligand Observed NMR Experiments
CPMG WaterLOGSY STD
Reference Spectrum – Fragment 1.5 mM
Fragment 1.5 mM + Target Protein 30 μM
Small molecules have a relatively long
relaxation time (T2: ~1 s).
Protein have a shorter relaxation time
(T2: ~1-50 ms).
100-400 ms delay introduced before
accquisition
Bulk water’s magnetization is
disturbed and transferred to:
- Free ligand in solution
- Ligand bound to the protein
NOE of opposite sign
Spectrum A: saturation pulse ( 0.5-1
ppm, methyl region). Protein-selective
resonances saturation and transfer to
bound ligand.
Spectrum B: saturation pulse off-
resonance (80 ppm).
In A only bound ligand is saturated – the
difference spectrum (B-A) will show
only the resonances of the bound ligandFragments that show binding in at least one
of these experiment is considered a hit.
Outcome of Three Fragment Screenings
Target protein Fragment library
Differential Scanning
Fluorimetry (DSF)
Bio-Layer
Interferometry (BLI)
1D Ligand Observed NMR
(CPMG, WaterLOGSY and STD)
Target :
A, B and C
Fragment library
(~1200)
DSF hit rate:
1.4%, 1.6% and 2%
BLI hit rate:
0.8%, 1.6% and 3.5%
Confirmed hits by NMR
62.5%, 69.2% and 33.3%
Analysis of BLI and DSF Performances as Screening Techniques
DSF17
DSF17
Target A
BLI10
BLI10
3
DSF19
DSF19
Target B
BLI20
BLI20
0
DSF24
DSF24
Target C
BLI42
BLI42
3
DSF and BLI show very little hit overlap
Running the screening in parallel with two orthogonal
techniques reduces the number of false negative hits
Analysis of BLI and DSF Performances as Screening Techniques
NMR Validate Hits for DSF
11 (64.7%)
16 (84.2%)
7 (29.2%)
11.3 (59.3%)
NMR Validate Hits for BLI
6 (60.0%)
11 (55.0%)
15 (35.7%)
10.6 (50.2%)
NMR validated overlapping hits: 2 (66.7%)
NMR validated overlapping hits: 1 (33.3%)
NMR validated overlapping hits: 1.5 (50.0%)
Conclusions
BLI is an effective tool to identify genuine fragment hits.
Current experimental setting allow to perform a single-point concentration
step on ~1200 fragments plus a follow up 6-point concentration step using 15
28h of time machine and < 0.5 mg of protein
NMR-Validated hits
10.6 (50.2%)
BLI and DSF have similar hit rates but BLI has a higher propensity to select false positives
Average
NMR-Validated hits
10.6 (50.2%)NMR-Validated hits
11.3 (59.3%)
To reduce false negative hits is critical to
perform the primary screen with two
orthogonal techniques
Acknowledgments
Compound
Management Team
Manuel Blank
David Robinson
(Drug Discovery Unit)
Emil Bulatov
Federica Cettorino
Martina Casale
Alessio Bortoluzzi
Alessio Ciulli Group
University of Dundee
22nd April 2015
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