screening bio ms - agilent bioms.pdf · screening bio ms lead generation ... success story: nhr...
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Screening Bio MS
Lead Generation – Candidate Realization
Sanofi R&D Toulouse
Aurélie VASSORT (Ph.D.)
Jacqueline BLOUIN
Pierre-Jean ROCKSTROH
Dominique SOUMEILLAN
2 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
Screening Bio MS: 2 unique & global activities
MS BIOPHYSICAL SCREENING
Global & unique support for S2H & H2L (MoA) Techno: 2-dimensional SEC-LC/MS
Success story: NHR (only screening method which provided validated hits)
Target druggability
Scre
en
assayab
ilit
y
Brute Force
HTS
HMTS has
high PoS
Develop
new
approaches
Improve
Readout
MS ENZYMATIC SCREENING
Global & unique support for S2H Techno: 2-dimensional SPE-MS/MS
Success stories: 2 enzymes (enabling screens)
1 SEC-LC/MS platform
2 SPE-MS/MS
platforms
3
Activity 1:
MS Biophysical Screening
Activity started in 2006
Platform built in-house from scratch
MTS started in 2008
4 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
Target protein
Pool of
compounds
+ 1. Incubation
Protein-ligand
complex
Unbounds
Size Exclusion Chromatography
2. Complex isolation
from unbounds 24s
53s
Collected
fraction:
complex
4. Data deconvolution
& ligand identification
Ligand identified
SN
OH
NO
O
NHO
Dedicated algorithm
3. Complex
dissociation
& detection
of ligand
Online high
resolution
UPLC/ESI-MS
0 8 min
+
Affinity Screening by Online SEC-LC/MS
5 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
1st dimension:
SEC
Sample
loop
2nd dimension:
RP-UPLC
04-Aug-2009 11:09:35LCT Premier
Time0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
6
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80A
U0.0
2.5
5.0
7.5
1.0e+1
Pka_Stauro_C2599D_04082009_07 3: Diode Array Range: 1.21e+10.87
0.41
Pka_Stauro_C2599D_04082009_07 HP1100 UVAn1
2.04e6
1.38
25s
Complex: collected in sample loop and sent to ESI-LC/MS
Unbound small molecules waste
SEC-UV before valve & sample loop
SEC-UV after valve & sample loop
52s
SEC-LC/MS Platform
6 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
●Screening collections already prepared by Chemical Library
● Pooling « plate by plate », 96-wplates format, max 200 cpds/well
● Sanofi Inner Core 50K: 4 plates (pools of 160 cpds/well)
● Sanofi Inner Core c100K: 4 plates (pools of 190 cpds/well)
● Sanofi Inner Core updates (57K): 4 plates (pools of 177 cpds/well)
● Natural Products (14.4K): 1 plate (pools of 180 cpds/well)
● Tucson (23K): 2 plates (pools of 140 cpds/well)
●Incubation mixtures
● Prepared in our lab (using PlateMate Plus)
● Restriction to use 96-wplates
●Data analysis
● Done in our lab
● Using a dedicated algorithm developed in-house
● Runs on Linux cluster 30 min for 50K analysis
Compounds & Collections
7 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
SCREENING: T2S, S2H
Large mixtures: 50-200 cpds - Instead of HTS - Parallel of HTS for orthogonal results
Primary screen for 50K (mixtures): - 4 days - 10 nmoles of protein (0.2 – 0.8 mg)
Confirmation screen (singles): - 100 cpds/day - 30 pmoles of protein / cpd (0.6 – 2.4 µg)
BACK-SCREENING: S2H
Small mixtures: 5-10 cpds - 500 cpds/day - 30 pmoles of protein / mixture
MoA STUDY: H2L, L2C
Single cpds
- To validate the chemical series (binding to the target) y/n + quantification of binding (%binding: bound/total)
- To determine the binding partner for PPI (idem)
- To determine the binding site: competition studies using ref cpds and/or btwn active cpds
- 100 cpds/day for binding - 30 pmoles of protein / cpd - 40 cpds/day for quantification - 60 pmoles of protein / cpd - 30 cpds/day for competition - 60 pmoles of protein/cpd
How SEC-LC/MS can support projects?
8 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
MS Biophysical Screening SWOT Analysis
Strength Weakness
● With respect to affinity screen:
●Tests all possible binding sites at once
● With respect to MS:
●Label-free technology
●Straightforward ID of the hits (from mixtures)
●Can screen unpurified compounds
●Sensitive: limited solubility issues (low conc)
● Throughput / cost:
●MTS: 15K / day
●Screening of mixtures (200 cpds max)
●Low protein consumption
●Almost no assay development
●Low cost assay
● With respect to affinity screen:
●False positives: non specific binding
biological validation is compulsory
●Need pure and soluble protein (X-Ray)
● With respect to MS:
●False negatives: non ionizable cpds &
irreversible binders
●Need to know the molecular formulae of cpds
(e.g. Risk Sharing collections not feasible)
Opportunity Threat
● Soluble proteins
● Investigation of PPI MoA
● Flexibility for screening (zymogen…)
● In-house & validated protein QC methods
● MS screening expertise
● Less informative biophysical technique but
higher throughput
● Need to do competition expts to discriminate
between specific & non-specific binding
9 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
●Target: nuclear hormone receptor
● Screening approaches run in parallel: HTS (TR-FRET assay), MS Biophysical Screen,
virtual screens (structure & ligand-based)
●MS Biophysical Screening
● Binding confirmation of ref cpds
● Screen of Sanofi innercore 100K + NP + Tucson (150K) 159 binders (0.1%)
● 24 active cpds in functional assay (7 agonists & 17 inverse agonists)
● Active, specific & selective cpds
● Good enrichment during Back-Screening: 28 new cpds
● None of the actives from the HTS FRET assay are “active specific” in the functional assay
●Input of MS Biophysical Screening
● Only source of hits now in H2L phase
● Same binding site as ref cpd
Demonstrated by MS biophysical MoA
Confirmed by crystallography
2011 Successfull Case Study: Summary
10 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
●Purity & stability: electrophoresis on
a chip (Agilent Bioanalyzer)
2011 Successfull Case Study: Protein QC
X-ray batch HTS batch 1 HTS batch 2 HTS batch 3
Purity 98.9% 69.4% 81.2% 82.5%
Conformational state Mainly dimeric,
monomeric as traces Aggregates Aggregates Aggregates
26.3 kDa, purity 98.9%
Markers
Marker
27.6 kDa : monomer
54.7 kDa : dimer
Aggregates
●Absolute molecular mass: SEC-Multi-
Angle Laser Light Scattering (MALLS)
Conclusion:
In-house protein QC needed
11
Activity 2:
MS Enzymatic Screening
Activity started in 2011
Agilent RapidFire® coupled to Thermo MS
2nd platform installed mid-2012
12 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
Enzymatic Screening by Online SPE-MS/MS
Enzyme
+
Substrate
+
Product Enzyme
1. Incubation
Compounds (1-10)
+
Product Compounds
Micro Solid Phase Extraction
2. De-salt & de-protein
Product
(Substrate) 3. Quantification of
product (and substrate)
ESI-MS/MS
Product
13 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
Agilent RapidFire® Technology
14 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
●Sample preparation & injection
● Vmin = 40 µL compatible with 384-wellplates but not 1536
● Use of ARP (already prepared) 384-wplates (nL distribution) prepared by
Chemical Library no intermediate plates needed
●SPE purification
● 6 different chemistries of cartridges
● Lifetime: 3000 - 10000 injections, depending on the batch
● Automatic column changer (for 20 hrs unattended runs)
●Software automation
● 2 softwares (RapidFire & MS)
● Barcode scanner
● Data analysis using the RF Integrator followed by a software developed in-house data format is
compatible with existing generic tools used in screening
●Medium throughput screening = Caliper
● 320 cpds / plate, plus 3-4 columns of controls
● Maximum: 55 min / plate, 22 plates / day (20 hrs)
● Minimum: 90 min / plate, 14 plates / day (21 hrs)
● 4.5 to 7k cpds / day if single screening
● 23 to 35k cpds / day if mixtures screening (10 cpds / well, each cpd appears twice)
SPE/MS/MS Platforms
15 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
MS Enzymatic Screening SWOT Analysis
Strength Weakness
● With respect to MS:
●Direct readout of enzymatic reaction
●Label-free technology (native substrate)
●Sensitive method: accurate % conversion
●Specific method: no interference (autofluorescence, cross-reactivity…)
●Screening of mixtures (10 cpds max)
● With respect to RapidFire:
●Fully automated
●Low operational costs
● Throughput / cost:
●MTS: 4.5-7K or 23-35K / day
●Low cost assay
● With respect to MS:
The substrate & product should:
●Be ionizable
●Have a unique molecular mass
●Have a different molecular mass (≥1 amu difference) not applicable to isomerase
Opportunity Threat
● Enabling technology
● Cheaper & higher quality assay results for selected assays
● In-house & validated protein QC methods
● MS screening expertise & LIT collab
● MTS only but 2 platforms
16 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
Enzymes screened in collaboration with LIT
Target Other
screens
Drawbacks Strategy Throughput Results
Dehydrogenase
Oncology Cambridge
LIT Strasbourg
Fluorescence
(HTS)
Assay
interferences
Secondary screen
to eliminate false
positives: 6000 in
duplicates
4.5K cpds / day Confirmation rate close
to 100%
Sulfotransferase
E2C Toulouse
LIT Toulouse
Fluorescence
Radiolabeled
Binding
2 steps, indirect
Radioactivity
Not enzymatic act.
Primary screen of
270K in singles
4.5K cpds / day 446 positives (0.17%)
Dose-responses on-
going
Quinolone synthase
Infectious Diseases TL
LIT Frankfort
Caliper Assay cost Primary screen of
384K in mixtures
+ 40K in singles
35K cpds / day M
7K cpds / day S
14.2K positives (3.3%)
4302 confirmed (1.0%)
3218 actives (0.8%)
Mono-oxygenase
Merial, Cambridge
LIT Strasbourg
Caliper Assay cost Secondary screen
to eliminate false
positives: 3200 in
duplicates
5.5K cpds / day On-going
Ceramide synthase
Diabetes, Frankfort
LIT Frankfort
Caliper Assay cost Primary screen Assay dev on-going
21 A. Vassort & coll. – Screening Bio MS – LGCR Toulouse
●LIT (assays)
● Frankfort team
• Ziyu Li
• Sonja Mueller
• Martina Sauerborn
• Heike Kohler
● Strasbourg team
• Walter Englaro
• Julia Frappier
• Nathalie Derimay
● Toulouse team
• Florence Pecceu
• Muriel Marion
• Christophe Pettereau
● AnSci Frankfurt (assays)
● Anja Pfenninger
● LIT-CL Toulouse (collections & plates logistics)
● Olivier Casamitjana
● Evelyne Gros
● Elodie Ferrer
● Carine Simonato
● Sébastien Issindou
● SDI (informatic tools)
● Olivier Stepien
● Luc Gauthier
Collaborations