RapidFire Selected Therapeutic Areas

and Screening Techniques

RapidFire: SPE/MS/MS

Life Sciences Group

William A. LaMarr, Ph.D.

Senior R&D Manager, RapidFire

RapidFire Mass Spectrometry

Ultra-fast autosampler &

online SPE system

• Replaces LC in LC/MS

• Reusable SPE cartridge

• Integrates with standard ESI MS

instruments

• Cycle time = 7-13 s/sample

Compatible with various matrices

• Sub-cellular fractions

• Cell culture supernatants

• Cell / tissue extracts

• Biological fluids

Advantages of mass spectrometry

• True label-free detection

• Direct, quantitative measurements

• Native reaction substrates & products

– (no radioactivity, no surrogate analytes,

no indirect or secondary components)

• Functional biochemical assays

– (rather than target binding assays)

instead of :

PO4

PO4

Limitations of MS

Molecules must be charged

Desalting step required

Sample purification is

– Serial

– slow

Instrumentation is expensive,

– not easily scalable

– to meet demand

Applications of the RapidFire Platform

1) Native Analyte Detection

- surrogate substrates can introduce confounding factors, effect enzyme kinetics, and produce data artifacts

2) Replace Intractable Assays

- assays may present challenges in workflow, may be resource intensive, may be cost prohibitive, may present regulatory issues (radioactivity)

3) Enable Target Classes

- multiple modification events on the same substrate are impossible to track by many common optical and radioactive methodlogies

ADS Customer Base

One

Target

Customers

Multiple

Target

Customers

Primary

Screening

(6x)

“Secondary”

Screening

(27x)

Large Pharma

Biopharma

Biotech

Secondary Screening &

Support

Primary Screening

“High

Profile”

Targets

“Unique”

Targets

Targeted Therapeutic Areas

Anti-Infectives

Inflammation

Metabolic Disorder Oncology

Cardiovascular

Disease

Inflammation

Other

Epigenetics

Metabolic

Disorder

Metabolic Disorder

Metabolic Disorders

Class of genetic disease that encompasses varied

conditions • Inborn errors of metabolism

• Congenital metabolic disease

Most are due to a single enzyme mutation effecting conversion of

substrate to product and often result in:

• accumulation of toxic substances

or

• reduced ability to synthesize essential compounds

Because of the diverse nature of the diseases in the group, accurate

numbers for incidence are difficult to determine. One Canadian study*

found that approximately 15% of single gene disorders in the population are

considered metabolic disorders.

* Applegarth DA, Toone JR, Lowry RB (January 2000). "Incidence of inborn errors of metabolism in

British Columbia, 1969-1996". Pediatrics 105 (1): e10

Enzymes and associated disorder(s)

An example of enzymes discussed in today’s

presentation:

Enzyme Role Associated Metabolic

Disorder(s)

Serine palmitoyltransferase Involved in sphingolipid biosynthesis Hereditary sensory neuropathy type 1

Acetyl CoA Carboxylase Involved in fatty acid synthesis Type 2 Diabetes

ATP citrate lyase Crucial in many biosynthetic pathways

including lipogenesis and cholesterolgenesis Fatty liver disease, type 2 diabetes

GM3 Synthase Involved in the biosynthesis of complex

gangliosides Infantile seizure/epilepsy disorder

Stearoyl CoA Desaturase Important for the desaturation of fatty

acids Obesity, liver disease

Diacylglycerol Acyltransferase Catalyzes the synthesis of triglycerides from

digylcerides obesity

Acetyl-Coenzyme A Carboxylase

Acetyl Coenzyme A

Malonyl Coenzyme A

KH14CO3

14C

O

OH2

+

AT

P

Acetyl-Coenzyme A

AssayConditions

50 mM HEPES, pH 7.4

2 mM MgCl2

2 mM tripotassium citrate

2 mM DTT

0.75 mg/mL BSA

4 mM ATP

12.5 mM KHCO3

20 mM Acetyl-Coenzyme A

*ACC assay conditions based on previously published 14C-incorporation assay

protocol: Harwood, H.J., Jr. et al. J. Biol. Chem. (2003), 278 (39): 37099-37111

Acetyl-Coenzyme A (m.w. 809)

Malonyl-Coenzyme A (m.w. 853)

* Harwood, H.J., Jr. et al. Curr. Opin. Investig. Drugs (2004), 5 (3):

283-289

* Sumper, M. Eur. J. Biochem. (1974), 49 (2):

469-475

Stearoyl-Coenzyme A Desaturase

OH

O-O-

HHN

O

HN

O

OP

OO

O OP

O

O-

P O OH

O

N

N

NH2

N

N

NH4+ NH4

+

NH4+

HO

O

S3H

3H

OH

O-O-

HHN

O

HN

O

OP

OO

O OP

O

O-

P O OH

O

N

N

NH2

N

N

NH4+ NH4

+

NH4+

HO

S

O

3H

O3H

+

Stearoyl-Coenzyme A

Oleoyl-Coenzyme A

* Soulard, P., et al. Analytica Chimica Acta (2008), 627 (1):

105-111

0.00

0.20

0.40

0.60

0.80

1.00

0 50 100 150 200 250 300 350 400 450 500 550

Plate Number

Z' S

co

re

> 200,000 wells screened

Diacylglycerol Acyltransferase

N

N

N

NH2

N

O

OP

OH

O

O

OPO

OH

OH

O

HN

HN

O

S

OHO

OH

PHO

O

O

Oleoyl-coenzyme A

O

O

O14

C14C

14C

OH

O

Diolein

O

O

O14

C14

C

14C

O

O

O

Triolein

IC50 = 49.1 nM

Z’ Score = 0.77

Epigenetics

ThioGlo Assay – SAH detection

Protein Methyltransferase Assay Strategies

AlphaScreen

Pan- methylation or

selective antibodies

are needed for multiple

states

F. Liu et.al., Journal of Medicinal Chemistry. 2009:669-81.

Masoud Vedadi

Amy Quinn

Protein Methyltransferase Assay Strategies

Transcreener Epigen

methyltransferase

assay principle.

Klink T A et al. J Biomol Screen 2011;17:59-70

Fluorescence

Polarization

Methylation-Sensitive Proteolysis Assay on EZ

Reader II

26

Wigle et al, Chemistry & Biology, 2010

Co-Product Measurements

-Histone Methyltransferases (HMTs)

S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH)

-Histone Acetyltransferases (HATs)

Acetyl-coenzyme A (ACoA) to Coenzyme A (CoA)

-Sirtuin Deacetylases (SIRTs)

Nicotinamide adenine dinucleotide (NAD+) to

nicotinamide + 2’-O-acetyl-ADP-ribose (OAADPr)

Direct measurement of the OAADPr product of sirtuin reactions

NN

N

NH2

N

O

OH OH

O P O

OH

O

P O

O-

O

O

OH OH

N+

NH2

O

NAD+

NN

N

NH2

N

O

OH OH

O P O

OH

O

P O

O-

O

O

OH O

OH

O

Acetylated

Protein

De-Acetylated

Protein

+

2’-O-acetyl-ADP-ribose

N

NH2

O

Nicotinamide

SIRT1, SIRT2, SIRT3 & SIRT5 & SIRT6?

SIRT1 - Enzyme Titration Timecourse

Reaction Conditions:

50 mM Tris pH 7.5

137 mM NaCl

2.7 mM KCl

1 mM MgCl2

0.05% BSA

5 mM DTT

100 mM NAD+

10 mM p53 peptide (Anaspec cat # 62121)

~12.5 minute analysis time

2’-O-acetyl-ADP-ribose

Internal Standard

SIRT1 - Enzymatic Parameters (Linearity, Km & IC50)

2’-O-acetyl-ADP-ribose Based Analysis

R² = 0.9995

0.0E+00

2.0E-05

4.0E-05

6.0E-05

8.0E-05

1.0E-04

1.2E-04

0 0.001 0.002 0.003Init

ial V

elo

cit

y (

V0)

Enzyme Dilution

Km = 28 ± 5 mM

Km = 54 ± 1 mM IC50 = 77 ± 1 mM

Comparison of Peptide Based and 2’-O-acetyl-

ADP-ribose Based Assay Parameters

Km of NAD+ Km of peptide IC50 of nicotinamide

SIRT1

SIRT3

SIRT2

Peptide

Read

2OAADPr

Read

Peptide

Read

2OAADPr

Read

Peptide

Read

2OAADPr

Read

38 ± 4 mM 54 ± 1 mM 25 ± 6 mM 28 ± 5 mM 62 ± 1 mM 77 ± 1 mM

46 ± 11 mM 50 ± 8 mM 8 ± 3 mM 12 ± 1 mM 10 ± 1 mM 11 ± 1 mM

118 ± 44 mM 144 ± 21 mM 4 ± 1 mM 6 ± 1 mM 31 ± 1 mM 39 ± 1 mM

Direct Peptide Measurements

Histone Demethylases

Lysine Demethylase 1 (LSD-1) uses Flavin Adenine Dinucleotide (FAD)

Jumanji Domain 2a (JMJD2a) uses Fe+2 mediated oxidative chemistry

Histone Deacetylases

Histone Deacetylase 1 (HDAC-1) uses a metal dependent hydrolysis

SIRT1 - Enzyme Titration Timecourse

Reaction Conditions:

50 mM Tris pH 7.5

137 mM NaCl

2.7 mM KCl

1 mM MgCl2

0.05% BSA

5 mM DTT

100 mM NAD+

10 mM p53 peptide (Anaspec cat # 62121)

~12.5 minute analysis time

0 minutes

240 minutes

180 minutes 120 minutes

60 minutes

45 minutes

30 minutes

15 minutes Enzyme

Deacetylated Peptide

Acetylated Peptide

SIRT1 - Enzyme Linearity

0

5

10

15

20

25

30

0 10 20 30 40 50 60

Perc

en

t C

on

vers

ion

Time (minutes)

0.00013 x enzyme

0.00067 x enzyme

0.0032 x enzyme

R² = 0.9999

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.001 0.002 0.003

Init

ial V

elo

cit

y (

V0)

Enzyme Dilution

Enzyme Titration / Timecourse Enzyme Linearity

SIRT1 - Enzymatic Parameters (Km, IC50, etc…)

Km = 25 ± 6 mM Km = 38 ± 4 mM

IC50 = 62 ± 1 mM

Deacetylated peptide

Acetylated peptide

Nicotinamide

~ 3 minute analysis time (8-point log-dilution IC50 curve, n=3)

Literature IC50 value ~ 50 mM Bitterman et. al., J. Biol. Chem. (2002) 277: 45099-45107

Marcotte et. al., Anal. Biochem. (2005) 332:90-99

SIRT2 - Enzymatic Parameters (Linearity, Km & IC50)

y = 104.22x + 0.0057 R² = 0.9925

0

0.05

0.1

0.15

0.2

0.25

0 0.001 0.002

Init

ial V

elo

cit

y

Enzyme Concentration (x stock)

IC50 = 9.6 ± 0.1

mM Km = 46 ± 11 mM

Km = 8 ± 2 mM

SIRT3 - Enzymatic Parameters (Linearity, Km & IC50)

IC50 = 31 mM

Km = 118 ± 44 mM

Km = 4.3 ± 1.0 mM

y = 63.717x - 0.0062 R² = 0.9998

0

0.05

0.1

0.15

0.2

0.25

0 0.001 0.002 0.003

Init

ial V

elo

cit

y

Enzyme Concentration (x …

Labeled vs. Un-labeled Sirtuin Assay

HN CH C

CH2

O

O

CH2

CH2

CH2

HN

ProteinProtein

O

H2N CH C

CH2

O

O

CH2

CH2

CH2

NH2

Protein

SIRT1/Chymotrypsin

“Labeled”

Acetylated

Peptide

De-Acetylated

Peptide

Resveratrol

Free fluorophore

Reaction

Activation

HN CH C

CH2

O

O

CH2

CH2

CH2

HN

ProteinProtein

O

HN CH C

CH2

O

O

CH2

CH2

CH2

NH2

ProteinProtein

“Unlabeled”

Acetylated

Peptide

De-Acetylated

Peptide

Resveratrol

X No

Reaction

Activation

SIRT1/Chymotrypsin

Howitz et. al., Nature (2003) 425:191-

196

Kaeberlein et. al., J. Biol. Chem. (2005) 280:17038-

17045

Beher et. al., Chem. Biol. Rug Des. (2009) 74:619-

624

Pacholec et. al., J. Biol. Chem. (2010) 285:8340-8351

SIRT1 - Substrate Dependant Activation by Resveratrol

“Labeled” Peptide “Unlabeled” Peptide

Rye et. al., J. Biomol. Screen. (2011) 16:1217-1226

Milne et. al., Nature (2007) 450:712-716

Multiple Modification Events on a Single Peptide

Preferred

acetylation

sites

potential

acetylation site

potential

acetylation site

-Human p53 (368-386)

-19-mer peptide

- Six potential acetylation sites

Rye et. al., J. Biomol. Screen. (2011) 16:1186-1195

Prives et. al., Nature (2007) 450:712-716

Direct Measurement of Modification Events on

Whole Histone Proteins

10-2 10-1 100 101 102 103 104

0

20

40

60

80

100

[Garcinol] (mM)

Perc

en

t o

f C

on

tro

l

AcCoA/CoA (QqQ)

Protein (Q-TOF)

Incorporation of a high-resolution (Q-TOF) mass spectrometer into the RapidFire workflow allows the substitution of whole proteins for representative peptide based sequences in a high-throughput screening compatible mode.

Rye et. al., J. Biomol. Screen. (2011) 16:1186-1195

Screening Histone Demethylases in a

Pharmaceutical Drug Discovery Setting

Plant et. al., J. Anal. Biochem. (2011) 419: 217-227

Hutchinson et. al., J. Biomol. Screen. (2012) 17: 39-48

Melanie Leveridge, MipTec 2011, oral presentation

Kruidenier et. al., Nature (2012) 488: 404-408

Conclusions

Mass spectrometry is an excellent tool for epigenetic target based drug discovery because if it’s ability to:

- Directly measure native, label-free peptides and generic reaction co-products

- Directly and independently measure multiple modification events on single substrates

- Directly measure modifications to whole protein substrates

Fragment Based

Drug Discovery

Fragment Based Drug Discovery

BACE-1 Assay: Fragment Based Screen

Cary Eclipse Fluorescence

Spectrophotometer

X

Assay Setup

Fluorescent Peptide

Fluorescence Plate Reader

Unlabeled Peptide

Mass Spectrometer

Fluorescent Peptide

Mass Spectrometer

Unlabeled Peptide

Fluorescence Plate Reader

Assay System Characterization

Unlabeled Substrate By MS (UMS)

Fluorescently Labeled Substrate by FS (FS)

Fluorescently Labeled Substrate by MS (LMS)

Initial Screening Results

UM

S

LMS

FS all 3

UMS 211 14 41 -

LMS 14 32 22 -

FS 41 22 122 -

all 3 - - - 8

UMS

LMS FS

Hits by Assay Format

Hits by Assay Format

Hits Observed by MS Only

Follow-up of selected hits confirmed that compound autofluorescence (AF) obscured several hits

in the FS data, including the most potent analyte. Titration of that compound revealed a

concentration-dependent increase in signal in the FS assay, suggesting AF, while the MS data were

consistent with a traditional inhibition curve.

Hits Observed with the Labeled Peptide Only

A second class of compounds was uncovered consisting of those molecules that appear as hits when the

labeled peptide is employed (as in the FS and LMS assays), but do not show inhibition when the more

native substrate is used (UMS). These results suggest that perhaps compounds exist that interfere with

the enzyme’s ability to bind the peptide carrying the bulky label but not with the tighter binding exhibited by

the enzyme for the unlabeled substrate, raising the possibility of misleading data being produced when

modified substrates are employed.

Hits Observed with the Unlabeled Peptide Only

Yet another class of inhibitors was detected in the unlabeled assay (UMS)

but was not found with the fluorescent peptide (FS or LMS). Because MS

eliminates the need for unnatural modification of substrates, it allows the

study of more biologically relevant molecules. These more realistic

substrates could reveal activities that are lost with modified peptides,

possibly due to altered binding, which in this case was clearly revealed by

the Km experiments.

Unlabeled Substrate By MS (UMS)

Fluorescently Labeled Substrate by MS (LMS)

Conclusions • Robust assays were developed for both a labeled and an unlabeled substrate of the BACE-1 enzyme.

• Screening a fragment library against both substrates using both detection methods produced three disparate hit sets.

• FS and MS produced different hit sets when used as complementary detection methods on the same samples. While some MS hits (including the most potent) were obscured by autofluorescence in the FS assay, this did not account for all of the differences between the methods.

• MS generated different hit sets for the labeled and the unlabeled peptide, underscoring the importance of substrate selection.

•Label-free screening by high-throughput MS has proven to be a valid method for conducting activity-based screens of fragment libraries that enables the study of more native molecules and is less susceptible to confounding factors, such as AF.

Target

Binding

Assays

Mass Spectrometry Approaches to Binding Assays

Questions?

Agilent Technologies

www.Agilent.com

William A. LaMarr, Ph.D.

Senior R&D Manager, RapidFire

william.lamarr@agilent.com

781-928-2718