hit identification strategy at merck: high troughput maldi-tof€¦ · semiconductor industry −...

Martin Hoffmann

Discovery & Development Technologies

Merck KGaA, Darmstadt Germany

ELRIG 2018

February 2018

Hit Identificationstrategy at Merck: High TroughputMALDI-TOF

HT MALDI-TOF | M Hoffmann | Feb 20182

MALDI-HTS@MerckOverview

Life SciencePerformance

MaterialsHealthcare

• A wide range of high-tech chemicals, such as:

− Liquid crystals and OLEDmaterials for displays & lighting

− Effect pigments for coatings and cosmetic products

− Specialty chemicals for the semiconductor industry

− Functional materials for solar panels

• Innovative tools and laboratory supplies for the life science industry that make research and biotech production better, faster and safer

• Broad and in-depth portfolio of 300,000 products

• Industry leading e-commerce platform, SigmaAldrich.com

• Award-winning innovation

• Prescription drugs and solutions to treat cancer, multiple sclerosis, infertility, cardiovascular and metabolic diseases

• Over-the-counter products for a healthy lifestyle

• Allergy products


MALDI-HTS@MerckOverview

Guiding our core research activities are our Translational Innovation Platforms (TIPs). As the cornerstone of our discovery efforts, the TIPs are focused on advancing our science through to first-in-man across our therapeutic areas.

Immuno-oncology ImmunologyOncology Global Health

• Oncogenic signaling

• Antibody-drug conjugates

• DNA damage and repair

• Tumor metabolism

• Tumor plasticity

• Suppressed anti-tumor

responses

• Remodeling the tumor

microenvironment

• Re-engaging the immune

system and increasing the

immunogenicity of tumors

• Researching novel

biomarkers

• Identifying novel drug

resistance pathways

• T- and B-cell biology

Innate immunity

• Immune tolerance

• Cartilage biology /

Repair mechanisms

• Schistosomiasis

• Malaria


MALDI-HTS@MerckMotivation• Direct quantification of substrate and product

• Eliminates need to develop specific antibodies or other costly labels

and indirect coupled assays

• Multiplexing

• Observation of previously undetectable reaction types

• Reduced reagent costs

• Bruker Rapiflex MALDI Pharma Pulse

• 10 kHz scanning

• 3D smartbeam laser

• easy to clean ion source

• handles 384 or 1536 spot MALDI targets

• 15-30 min per target (>60 min with Ultraflex)

HTStec Ltd Market Report 2016


MALDI-HTS@MerckSet-up

Reaction in MTP

Addition ofmatrix

solution

Spotting on MALDI target

MeasureMALDI MS

Data Processing

• full automated HTS workflow & setup

• integration of MALDI MS in existing HTS system

• MALDI MS as new read-out alternative


MALDI-HTS@MerckWorkflow

Reaction in MTPAdd Matrix

solutionSpotting on

MALDI targetMeasure MALDI

MSData Processing

• assay format 384 or 1536 well plates

• plate handling by Scara robot

• disposible MALDI targets

• dispensing reagents by Multidrop combi

• incubation by Thermo Cytomat

• dispensing of matrix solution is challenging

• high organic solvent content

• matrix solution tends to crystallize easily

• Multidrop Nano dispenser as best option

• costum made teflon tubing kit with in-line filter




solutionSpotting on


MSData Processing

384 well spotting

• with Beckman Biomek FX

• disposable tips

• 1-2 µL spot volume

1536 well spotting

• with Analytik Jena CyBio Well

• non-disposable ceramic tips

• reservoir available

• 200-500 nL spot volume

1536 well spotting

• with Labcyte ECHO

• ultrasonic liquid transfer

• 50-100 nL spot volume

• narrow working window

1536 well spotting

• PinTool & Beckman Biomek FX

• stainless steel pins

• spot volume not exactly controllable

• often missing spots




solutionSpotting on


MSData Processing

Automated Measurement:

• Beckman SAMI Scheduler

• Assay run controlled by SAMI

• MALDI run controlled by MPP

• AutoXecute protocols used

Data Analysis:

• Online: Bruker MPP

• Genedata Screener

MALDI

Pharma PulseFlexControl

(AutoXecute)

SAMIMALDI

Pharma Pulse

Amplius

Module


MALDI-HTS@MerckcMET

• cMET kinase as method evaluation study

• cMET is a tyrosine kinase receptor

• activating ligand: Hepatocyte growth factor HGF

• important in oncogenic signaling and angiogenesis

• overexpressed in certain cancers

• associated with tumor growth and tumor angiogenesis

• Screening for cMET kinase inhibitors

• cMET 956-1390 fragment by ProQinase (79 kDa)

• 14 aa peptide as artificial substrate

A. Zhang et al., Bioorg. Med. Chem. Lett. 2012, 22, 6368-6372.


Srctide: NH2-GEEPLYWSFPAKKK-CONH2FITC-Srctide: FITC-linker-RGEEPLYWSFPAKKK-CONH2

Reference method:

Capillary electrophoresis (EZ Reader)

• requires peptide with fluorescent tag

• chromatography with fluorescence detector

• expensive and error prone chips

MALDI Mass Spectrometry:

Bruker Rapiflex

• no tag required

• mass spectrum in 800-3000 Da region

• substrate/product peak area ratio for calculation

conversion% =area product ∗ factor

area substrate + area product ∗ factor

conversion% =area product

area substrate + area product

MALDI-HTS@MerckcMET

Dm/z


MALDI-HTS@MerckcMET

Buffer & Matrix Selection Challenges:

• Assay Development has to adapted for MALDI HTS

• common assay conditions are not suitable

• high buffer and salt concentration suppress ionization

• detergents suppress ionization and overlay spectrum

• but: buffer still has to support enzymatic reaction

Solutions:

• lower buffer concentration

• low Mg concentration (required for enzymatic reaction)

• low detergent (required for enzymatic reaction and dispensing pipes)

Results:

• buffer: 1 mM HEPES, 1 mM MgCl2, 2 mM DTT, 0.01% Brij-35

• matrix mixture: 4-HCCA in 80% MeCN/H2O with 2% TFA

• mixed with assay in 1:1 ratio

5 m

M M

gC

l2

3 m

M M

gC

l2

1 m

M M

gC

l2

5 m

M M

gC

l2

3 m

M M

gC

l2

1 m

M M

gC

l2

5 m

M M

gC

l2

0

1 0

2 0

3 0

4 0

5 0

C o m b in e d c M e t A s s a y A d ju s tm e n t

Co

nv

ers

ion

[%

]

2 0 m M H e p e s /

0 .0 1 % B r ij-3 5

1 m M H e p e s /

0 .0 1 % B r ij-3 5

2 0 m M H e p e s /

0 .0 1 % T r ito n X -1 0 0

J.Chandler, C. Haslam, N. Hardy, M. Levenridge, P. Marshall, SLAS Discovery 2017, 22, 1-8.

K. Beeman, J. Baumgärtner, M. Laubenheimer, K. Hergesell, M. Hoffmann, U. Pehl, F. Fischer, J.-

C. Pieck, SLAS Discovery 2017, 22, 1203-1210.

Marshall, SLAS Discovery 2017


MALDI-HTS@MerckcMET

MALDI

Caliper

A C

D

B

Compound ID

IC50 [

M]

Caliper

-IC

50 [

M]

Maldi - IC50 [M]

Compound-C

Compound-M

Compound-F

Compound-C

Compound-M

Compound-F

K. Beeman, J. Baumgärtner, M. Laubenheimer, K. Hergesell, M. Hoffmann,

U. Pehl, F. Fischer, J.-C. Pieck, SLAS Discovery 2017, 22, 1203-1210.


MALDI-HTS@MerckCovalent Binders

• Covalent Binder Screening

• probing small molecule – protein interactions

• compounds with reactive groups (warheads)

• protein target with reactive amino acid in active pocket

• binding of compound in active pocket leads to covalent bond

• approx. 1000 compound library



9666.8

19336.8

19767.09883.6

19970.6

A1123429 0:A12 MS

0

2

4

6

8

4x10

Inte

ns. [a

.u.]

9897.1

19796.5

A1123429 0:A1 MS

0.0

0.5

1.0

1.5

4x10

Inte

ns. [a

.u.]

9774.9

19553.3

A1123429 0:A5 MS

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [a

.u.]

10000 12000 14000 16000 18000 20000m/z

• Covalent Binder Screening

• 384 well format

• MALDI linear positive mode

• mass shift of protein

• target engagement = conversion%

• calculated on peak intensity

conversion% =intensity product

intensity substrate + intensity product

unbound target (0%)

reference compound (100%)

medium binding compound (62%)



• Method of choice: LC-ESI MS

• reversed phase column sample clean-up

• high resolution

• charge state detection -> deconvolution needed

• approx. 30 min per run

• MALDI TOF as high troughput alternative

• dilution of assay mix with matrix

• direct observation of full length protein

• only two charge states observed

• approx. 30 s per run

• good overall correlation

• weaker correlation at low/high conversions

• protein size is limited due to resolution

Correlation

ESI conversion%

MA

LD

I co

nvers

ion

%

0.0 0.2 0.4 0.6 0.8 1.00.0

0.2

0.4

0.6

0.8

1.0

𝑦 = 1,24 ± 0,08𝑥 − 0,07 ± 0,03 (𝑅2 = 0.74)


MALDI-HTS@MerckOutlook

DNA Damage & Repair

Strategic Focus of TIP Oncology @ Merck

• DNA repair pathways are targets for cancer therapy

• Treatment option for resistent cancer cell lines

• Reactions are difficult to monitor with fluorescent labels

• Common gel based assays are not applicable for HTS

• MALDI MS read-out as alternative

Base Excision Repair Pathway

single strand base damage

Glycosylase

abasic site

AP Endonuclease

dRP Lyase

Polymerase


Dr. Martin Hoffmann

Merck KGaA Healthcare

RD-XPA

A31/130

Frankfurter Str. 250

64293 Darmstadt / Germany

[email protected]

RD-XPA

Thank you for your attention!

hit identification strategy at merck: high troughput maldi-tof€¦ · semiconductor industry −...

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