Screening Bio MS - Agilent BioMS.pdf · Screening Bio MS Lead Generation ... Success story: NHR (only screening method which provided validated hits) ... 2011 Successfull Case Study:

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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


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


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


●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


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?


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)


●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


●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


●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


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


Agilent RapidFire® Technology


●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


MS Enzymatic Screening SWOT Analysis

Strength Weakness


●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


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


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


●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

Screening Bio MS - Agilent BioMS.pdf · Screening Bio MS Lead Generation ... Success story: NHR (only screening method which provided validated hits) ... 2011 Successfull Case Study:

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