user’s meeting, brussels, november 2016 - bruker · user’s meeting, brussels, november 2016...
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Dr. Till Kühn
VP Applications Development MRS, Bruker BioSpion
User’s meeting, Brussels, November 2016
Fragment-based drug discovery
Innovation with Integrity
The principle of Fragment Based Screening
from efficient fragments to Drug candidates
December 5, 2016 2
Fragment high “ligand efficiency” (LE), medium IC50
Candidate high LE, low IC50
0.91mM
0.59
0.07mM
0.55
5.9nM
0.49
3.0nM
0.42
IC50:
LE:
Grow, optimize
Advantages of FBS:
Fragment chemical space (109) << drug like chemical space (1030)
• Fragment space easier to explore
• Smaller libraries with higher diversity (~ 1’000 – 5’000 compounds)
• Higher hit rates (0.1% - 10%)
NMR in Fragment based Screening
More than one answer possible
% respondents using NMR technique
Summary:
• FBS is used more and more often
• NMR is one of the top 2 methods
• People use more than one technique
• Ligand and Protein detected NMR used
December 5, 2016 4
Fragment Based Screening
NMR vs. SPR
NMR Fragment Screening SPR Fragment Screening
100 samples, 500-1000 compounds per day (19F 3000 compounds/day) (5-10 compounds per sample)
500 compounds per day, single point measurement (depends on instrument)
Operational costs: ~45k per year (96 well format NMR tubes, cryogens and service contract)
Operational costs: 45-50k per year (chips for target immobilization, consumables, solvents and service)
QC of fragments possible as part of process, inherent concentration information aids hit validation
No QC of fragments possible during process, independent QC required
No issue, one tube per sample: bad sample does not stop the screen
Sticky compounds may dismantle the chip during screening
Clumsy, home-built acquisition automation, data organization, no workflow support
Easy to use interface for operation, data analysis and export
Throughput
Running costs
Data Quality
Bad samples
Data Analysis
Ligand observed methods
identify binders from mixtures
Protein observed methods identifiy
binders and binding site on target
- NO isotopic labeling of the protein
- NO size limitation of the target
- Little amounts of protein needed (4-10
mgs)
- Little amounts of ligands needed (0.025
– 0.250 mM in each sample)
- Binding site on target can be identified
- Isotopic protein labeling required (13C or 15N)
- Size limitation of target
- Amount of protein required depends on
application
Two NMR Screening principles
Ligand observed methods
identify binders from mixtures
Protein observed methods identifiy
binders and binding site on target
No crystal structure available
Binding site in crystal contact (often for
PPI targets)
- NO isotopic labeling of the protein
- NO size limitation of the target
- Little amounts of protein needed (4-10
mgs)
- Little amounts of ligands needed (0.025
– 0.250 mM in each sample)
- Binding site on target can be identified
- Isotopic protein labeling required (13C or 15N)
- Size limitation of target
- Amount of protein required depends on
application
Two NMR Screening principles
Excess of ligand (i.e. 0.25 mM)
small molecule, different NMR properties than protein
NMR Fragment Based Screening
The concept
Low protein concentration (i.e. 0.01mM)
Large molecule with distinct NMR properties
High ligand concentration, low protein concentration
Ligand in contact with protein (=binding) adopt it’s physical properties during residence time
NMR Fragment Based Screening
The concept
Excess of ligand (i.e. 0.25 mM)
small molecule, different NMR properties than protein
Low protein concentration (i.e. 0.01mM)
Large molecule with distinct NMR properties
norm
alized inte
nsity
time
T2
NMR Fragment Based Screening
The concept
Binding ligand keeps properties when back in the pool, other ligands are unchanged
norm
alized inte
nsity
T2
time
Excursion: Fluorine Fragment Screening
5’-19F-Tryptophan, H2O/D2O, phosphate buffer, pH 7.4
5’-19F-Tryptophan
+ Human Serum Albumin, H2O/D2O, phosphate buffer, pH 7.4
20 ms relaxation delay
200 ms relaxation delay
20 ms relaxation delay
200 ms relaxation delay
Spin-Echo pulse program for
relaxation measurement
binding fragments: faster relaxation when compared to non-binders
Advantages of 19F screening:
• No water suppression
• Usually one peak per fragment
• No buffer signals
• Up to 30 fragments per mixture
• Lower compound and protein concentration
⇒ Increased throughput, higher efficiency
Excursion: Fluorine Fragment Screening
typical conditions:
protein = 8 µM
compound = 20 µM
sample volume = 170 µL
(3mm tube)
measuring time = 8 min
600 MHz QCI-F CryoProbe
Courtesy of Dr. M. Blommers, Novartis Pharma, Switzerland
Excursion: Fluorine Fragment Screening
Problem: Large Spectral Width
Phasing difficult if hard 180°pulses are used
Excursion: Fluorine Fragment Screening
Spin-Echo pulse program for
relaxation measurement
WHAT WORKS @ 700MHz:
1.5ms Crp90.comp4
Power of 13 us 90-square (19.5kHz)
Excitation profile 70kHz
= ca. 110ppm 19F @ 700MHz
Solution: broadband adiabatic refocusing pulse
Excursion: Fluorine Fragment Screening
WHAT WORKS @ 700MHz:
1.5ms Crp90.comp4
Power of 13 us 90-square (19.5kHz)
Excitation profile 70kHz
= ca. 110ppm 19F @ 700MHz
Spin-Echo pulse program for
relaxation measurement
Solution: broadband adiabatic refocusing pulse
Excursion: Fluorine Fragment Screening
1H decoupling significantly improves signal to noise
With 1H decoupling
Without 1H decoupling
Benefits of 1H decoupling
Excursion: Fluorine Fragment Screening
CryoProbe QCIF
19F-observe
1H-decouple
19F S/N 6000:1 @ 600MHz
Smart probe BBFO
19F-observe,
1H-decouple
19F S/N 565:1 @ 600MHz
N2 cooled CryoProbe Prodigy TCI
19F-observe without 1H-decoupling
19F S/N 2400:1 @ 600MHz
Probe options
Excursion: Fluorine Fragment Screening
Three Basic 1H NMR Experiments in FBS
Saturation Transfer Difference (STD)
Binders show up in difference spectrum, non binders don’t
Water-LOGSY
Binders have opposite phase to non-binders
T2/T1rho
Binders show strong attenuation
NMR FBS in practice
You mix several ligands per sample with protein:
• for higher sample throughput (ligands per sample) AND
• to identify binding by comparison to non-binder (qualitative aspect)
⇒ You need a reference spectrum of each individual compound
to make the connection to the actual binding molecule
STD
STD reference
Not binding
Weakly binding
Strongly binding
NMR FBS in practice
STD
STD reference
You mix several ligands per sample with protein:
• for higher sample throughput (ligands per sample) AND
• to identify binding by comparison to non-binder (qualitative aspect)
⇒ You need a reference spectrum of each individual compound
to make the connection to the actual binding molecule
NMR FBS in practice
STD
STD reference
You mix several ligands per sample with protein:
• for higher sample throughput (ligands per sample) AND
• to identify binding by comparison to non-binder (qualitative aspect)
⇒ You need a reference spectrum of each individual compound
to make the connection to the actual binding molecule
1. Before screening against any protein, you run each compound of library
2. Then make cocktails
3. Start screening campaigns
December 5, 2016 23
• Simple batch setup for the complete library
Setup
• Optimized experiments (auto water suppression etc.)
for master stock solutions (DMSO) for ligands in buffer (screening reference)
Automation
• Fully automated analysis on-the-fly or batch:
• integrity, concentration, purity, solubility
Analysis
• All information at a glance
• Easy cross check and manual editing
• Spread sheets to sort out bad compounds that don’t go into the mixtures
Results
Screening preparation & library QC on-the-fly
CMC-q sets-up, runs and analyzes your references
December 5, 2016 24
Screening preparation & library QC on-the-fly
It actually makes sense to check!
Our libraries had ca. 30% bad samples!
• 20% “no compound” no compound in stock solution, or not soluble in buffer
• 10% decayed or wrong compound
• 50% concentration off by more than +/-30%
• QC of DMSO stock solution
• QC and reference in buffer
• The actual protein screening
Tools for the practical work
New temperature control option in SampleJet
Individual temperature control for each of the 5 96 sample racks
The actual screening campaign
Data acquisition
1. Preparation: run & analyze data for single compound reference (e.g. CMC-q)
2. Mix compound cocktails, document in excel table
3. Run screening campaigns
• Parameter sets to automatically setup and optimize screening experiments
• Setup batch run in IconNMR
The actual screening campaign
Data analysis and interpretation – up to now
For each sample, you
• Open the STD spectrum
• Open the STD reference
• Open the Water-LOGSY
• Open the T2 experiment
• Open the T2 reference
• Scale all of them for amplitude
• Search for, and open all single compound reference spectra for that mixture
Before you can start analyzing
The actual screening campaign
Data analysis and interpretation – TS3.5 next pl
With TopSpin 3.5, next pl, you …
• Point to mixture table
• Indicate directory for single compound reference
• Indicate parent blank screening data (if applicable)
…. start analyzing!
December 5, 2016 29
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 30
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 31
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
Customizable Spectra Types by unique identifier
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
Customizable display layout
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 34
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 35
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 36
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
Customizable report layout
New FBS tool in Topspin
Data analysis and interpretation – TS3.5 next pl
December 5, 2016 38
Summary
New tools & experiments for FBS
• CMC-q to easily run and analyze single compounds in buffer for reference AND do QC on the fly (in buffer to test solubility!)
• New Parameter sets in TS3.5 pl6 to run optimized FBS experiments in full automation
• New SampleJet temperature control option to run DMSO samples in one plate, cooled protein samples in the other
• New tools in IconNMR to setup batches of screening samples quickly from customized Excel spread sheets
• Brand new FBS tool in TopSpin to help you quickly organize data, analyze it your way and keep track of your results reporting into configurable excel spread sheets
December 5, 2016 39
Alavar Gossert, Wolfgang Jahnke, Marcel Blommers, Cesar Fernandez, Paul Erbel
Daniel Wyss and Hugh Eaton
Stefan Jehle, Pavel Kessler, Fabrice Moriaud, Matteo Penestri, Anna Codina
Bettina Elshorst
Markus Schade
© Copyright Bruker Corporation. All rights reserved.
Innovation with Integrity
Copyright © 2011 Bruker Corporation. All rights reserved. www.bruker.com
Thank you for your attention
… and come see us for demos and discusions
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