with ip-one assay - cisbio · case study: hts of gpr-54 with ip-one assay marcie glicksman, phd...

Laboratory for Drug Discovery in

Neurodegeneration

Harvard Center for Neurodegeneration and Repair

Assays and Cellular Targets October 31, 2006

Case Study: HTS of Case Study: HTS of GPRGPR--5454with IPwith IP--One AssayOne Assay

Marcie Glicksman, PhDMarcie Glicksman, PhDSenior Director of Leads DiscoverySenior Director of Leads Discovery

Laboratory for Drug Discovery in NeurodegenerationLaboratory for Drug Discovery in NeurodegenerationBrigham and WomenBrigham and Women’’s Hospital & Harvard Medical Schools Hospital & Harvard Medical School


Neurodegeneration

OutlineOutline

• What is LDDN?• GPR54 Background• Why IP-One• HTS protocol• Summary


Neurodegeneration

Laboratory for Drug Discovery in NeurodegenerationLaboratory for Drug Discovery in Neurodegeneration

Mission of the LDDN is to ...

Create a new model for drug discovery that integrates the best of industry and academics.

Discover chemical agents that can be used as lead structures in the development of drugs to treat neurodegenerative diseases.


Neurodegeneration

Laboratory for Drug Discovery in NeurodegenerationLaboratory for Drug Discovery in Neurodegeneration

� Hypothesis-driven, screening-based approach.

� Managed by industry-seasoned professionals.

� Programs based on tight academic collaborations.

� Resourced for success – Commercialization of disease modifying therapeutics.

� Cover a broad range of targets in neurodegeneration.

� Forward-expansion – New collaborators and new disease areas.

Features of the LDDN model …


Neurodegeneration

GPR54: Case Study for LDDNGPR54: Case Study for LDDN


Neurodegeneration

The HypothalamicThe Hypothalamic--PituitaryPituitary--Gonadal AxisGonadal Axis

(-) GABA, OpioidsAspartate, Leptin, Dopamine, Glutamate, (+)NE, NPY, KisspeptinKisspeptin

Hypothalamus

AnteriorPituitary

Gonad

GnRH (+)

LH (+)FSH (+) Sex steroids (+/-)

Inhibin (-)

Activin (+)Follistatin (-)


Neurodegeneration

AIMAIMAIM

To identify GPR54 agonists, antagonists, and To identify GPR54 agonists, antagonists, and enhancers for therapeutic and research uses.enhancers for therapeutic and research uses.

hormone-dependent cancers, precocious puberty

Agonists:

Antagonists:

delayed puberty, infertility


Neurodegeneration

GPR54GPR54

kisspeptinkisspeptin

PLCß

PIPPIP22 DAG + IPDAG + IP33

GPR54, A G Protein-Coupled ReceptorGPR54, A G ProteinGPR54, A G Protein--Coupled ReceptorCoupled Receptor

• GPR54 is expressed primarily in brain, pituitary, and placenta.

Gq/11


Neurodegeneration

GPR54GPR54


PLCß




• Natural ligand of GPR54 = kisspeptin, encoded by gene KiSS-1

Gq/11


Neurodegeneration

GPR54GPR54


PLCß





• KiSS-1 is expressed in hypothalamus and placenta.

Gq/11


Neurodegeneration

GPR54GPR54


PLCß


GPR54, A G Protein-Coupled ReceptorGPR54, A G ProteinGPR54, A G Protein--Coupled ReceptorCoupled Receptor• GPR54 is expressed primarily in brain,

pituitary, and placenta.


• KiSS-1 is expressed in hypothalamus and placenta.

• Kisspeptin-54 was named metastin for its ability to inhibit tumor metastasis.

• GPR54 knockouts do not undergo puberty and antibodies will block LH surge

Gq/11


Neurodegeneration

GPR54GPR54


Gq/11

CaCa2+2+

CaCa2+2+PLCß


ER

RafRaf

MEK 1/2MEK 1/2

ERK 1/2

GonadotropinGonadotropin--releasing hormone secretionreleasing hormone secretion

PKC

GPR54-Coupled Signal Transduction PathwaysGPR54GPR54--Coupled Signal Transduction PathwaysCoupled Signal Transduction Pathways

??

IPIP22

IPIP11


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Agonists and enhancers of GPR54 will promote GnRHsecretion, while GPR54 antagonists will suppress GnRH

secretion.

HYPOTHESISHYPOTHESISHYPOTHESIS

GnRH NeuronGnRH Neuron

PituitaryPituitary

GPR54GPR54

GnRHGnRH

AgonistAgonist

XXX Antagonist

Reagents that modify GPR54 signal transduction and hence GnRH secretion will have wide-ranging applications in the fields of reproductive medicine and cancer therapy.


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Generation of Stably Transfected GPR54 Cell LinesGeneration of Stably Transfected GPR54 Cell Lines

Full-lengthhGPR54

cDNA

�� Chinese hamster ovary Chinese hamster ovary (CHO) cell line(CHO) cell line

•• Used in other published Used in other published GPR54 stable cell linesGPR54 stable cell lines•• Growth pattern simplifies Growth pattern simplifies clone selectionclone selection

�� Construct backbone Construct backbone ––pIRESneo3 (pIRESneo3 (ClontechClontech))

•• BicistronicBicistronic vector vector –– gene of gene of interest and selection marker are interest and selection marker are under control of same promoter, under control of same promoter, favoring selection of clones with favoring selection of clones with higher expression of higher expression of transgenetransgene


Neurodegeneration

Generation of Stably Transfected GPR54 Cell LinesGeneration of Stably Transfected GPR54 Cell Lines

28S

18S

P2 GPR

54-1

28S

18S

P2 GPR

54-1

Empt

yve

ctor

line

GPR

54lin

e

3 kb3 kb

Full-lengthhGPR54

cDNA


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Gs Gi Gq

cAMP � cAMP � IP3 �

Ca2+ �

GPR54 GPR54 isis a a GqGq coupledcoupled GPCRGPCR


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GqGq coupledcoupled GPCR GPCR signallingsignalling pathwaypathway

�q�� PLC

PIP2

OHOHHO

HOHO

OPO3H

OHOHH2PO3O

H2PO3OHO

OPO3H2

OHOHHO

HOHO

OH

IP1

Myo-inositol

Inositol

cascade

IP2

IP3

E.R.

Ca2+

Inhibitionby LiCl

IP1

In presence of LiCl,IP1 accumulation

upon GPCR activation

OPO3H2HO

HOH2O3P O

OHOH

Fluorescentsensing

IP3

IP3 lifetimeis very short

Radioactive detection


Neurodegeneration

• Did not have access to a FLIPR for measuring Ca++ flux

• Miniaturization to 384-well format to reduce reagent consumption

• Minimal number of steps to facilitate automation and maximize speed and efficiency

• Non-radioactive detection for throughput, safety, and waste disposal considerations

• Must have satisfactory sensitivity, accuracy, and reproducibility (Z factor)

Why Choose IP-One for HTSWhy Choose IPWhy Choose IP--One for HTSOne for HTS


Neurodegeneration

Homogeneous Time Resolved FluorescenceHomogeneous Time Resolved Fluorescence• HTRF® is a technology based on TR-FRET, a

combination of FRET chemistry and the use of fluorophores with long emission half-lives.

• HTRF uses lanthanide with an extremely long half-life (Europium), conjugation of Eu3+ to cryptate, an entity which confers increased assay stability

• Use of a ratiometric measurement that allows correction for quenching and sample interferences.

– Simplified assay miniaturization – Tolerant of additives e.g. DMSO & EDTA

– Cell-based functional assay


Neurodegeneration

• Competitive immunoassay with cryptate-labeled anti-IP1 monoclonal antibody and d2-labeled IP1. • LiCl is used causing the accumulation of IP1 upon receptor activation. • The assay can be run in a single microplate and requires only a single 1 hour incubation following cell stimulation.• No cross-reactivity with 50µM of (phospho) inositides phosphates

The IPThe IP--One One assayassay


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Kisspeptin StimulationKisspeptinKisspeptin StimulationStimulation

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -300

10

20

30

40

empty vector

GPR54

hkiss-10 (log M)

(CPM

/mg

prot

ein)

Tota

lIP

Prod

uctio

n�� KisspeptinKisspeptin Stimulates IP Accumulation In a DoseStimulates IP Accumulation In a Dose--Dependent Dependent

Manner in GPR54 Stably Transfected CellsManner in GPR54 Stably Transfected Cells


Neurodegeneration

96 96 1/2 384 384 sv

Totalvolume (µl)

Cellnumber 80,000 40,000 15,000 to

30,0008,000

100 50 20 to 40 10

IP-One assay miniaturization

Plate type


Neurodegeneration

IPIP--One One assayassay protocolprotocol

30,000 cells/40ul/well in white Nunc 384-plates

Seal with aeroSeals

Remove media

(add Kisspeptin for antagonist screen)

Add compound together with stimulation buffer

Add IP-One kit D2

Add IP-One Cryptatereagent

Seal with aeroSeals

Seal with aluminum seals


Neurodegeneration

IPIP--One One assayassay protocolprotocol

30,000 cells/40ul/well in white Nunc 384-plates

Seal with aeroSeals

Remove media

(add Kisspeptin for antagonist screen)

Add compound together with stimulation buffer

Add IP-One kit D2

Add IP-One Cryptatereagent

Seal with aeroSeals

Seal with aluminum seals

1 h-24 h


Neurodegeneration

Robotics Used for the ScreenRobotics Used for the Screen

• Beckman FX core system

• BMG PHERAstar or PerkinElmer Envision

• High energy xenon flashlamp excitation

• Simultaneous dual wavelength measurement for HTRF

• Integrated into robotic system


Neurodegeneration

Compound Library (110,000 compounds)Compound Library (110,000 compounds)� Computational filters applied to select compounds with an increased probability of oral bio-availability and blood brain barrier penetration

1670 FDA-approved drugs from Prestwick480 purified natural products4,100 N-Ac-tetrapeptide amides1570 compounds from academic organic chemists 800 proprietary compounds synthesized by LDDN

• All small molecules adhere to Lipinski’s rules.• Low proportion of toxicophores• Low proportion of unwanted functionalities.• Maximization of molecular diversity.

3000 Peakdale7000 Bionet 5000 CEREP 16,000 Maybridge50,000 ChemDiv6000 Enamine6000 IF Lab

� Plus . . . Program to collect additional compounds from academia.� Additional compounds being purchased.


Neurodegeneration

Control for Automation Control for Automation Control for Automation

EC501.25 x 10-9

5.61 x 10-10

1.03 x 10-9

-11 -10 -9 -8 -7 -6

0

25

50

75

100human kiss-10 #1human kiss-10 #2mouse kiss-10

hkiss-10 (log M)

%Ac

tivat

ion

�� % Activation with % Activation with KisspeptinKisspeptin DoseDose--ResponseResponse


Neurodegeneration

GPR-54 Z' Score for 120384-well Plates

0 20 40 60 80 100 1200.0

0.2

0.4

0.6

0.8

1.0

0.76 + 0.07(Mean+SD)

Plate Number

Z'Sc

ore

Coefficient of VariationPositive Control

0 25 50 75 100 1250

5

10

15

20

25

30

35

40

Plate Number

%C

V

Coefficient of VarianceDMSO Controls

0 25 50 75 100 1250

5

10

15

20

25

30

35

40

Plate Number

%C

V

Screening StatisticsScreening Statistics

Maximal kisspeptin DMSO

Z’ = 0.7S:B = 4-5


Neurodegeneration

GPR54 LineGPR54 Line

0

10

20

30

Ø kiss-1010-10 M

Fold

incr

ease

inIP

LDN-21810

kisspeptin 10-10 M

�� LDNLDN--21810 Stimulates IP Production in a GPR5421810 Stimulates IP Production in a GPR54--Specific and Specific and DoseDose--Dependent MannerDependent Manner

Empty Vector LineEmpty Vector Line

LDN-2181010-6 M 10-5 M 10-4 M 10-3 M

0

10

20

30

Ø 10-4 M 10-3 M kiss-1010-10M

Fold

incr

ease

inIP

LDN-21810

LDNLDN--21810 In Follow21810 In Follow--up Assaysup Assays


Neurodegeneration

Agonist Screening ResultsAgonist Screening Results

• The LDDN small molecule library of 110,000 compounds has been screened for GPR54 agonists.

• Currently characterizing one of the hits– LDN-21810.

• Unfortunately, HTS did not identify many hits. This is not surprising with peptide-ligand receptors.


Neurodegeneration

Strategy for Antagonist ScreeningStrategy for Antagonist Screening

• The PLC inhibitor U-73122 used as a positive control.

• Negative controls: kisspeptin without U-73122 and no D2 wells

• Currently undergoing optimization


Neurodegeneration

Radioactive IP assay with Radioactive IP assay with kisspeptinkisspeptin and Uand U--73122 73122 (antagonist)(antagonist)

0

5

10

15

20

25

30

35

40

notre

atmen

tan

tagon

ist10

-5M

+kis

s 10-9

Man

tagon

ist1.8

x 10-5

M+ kis

s 10-9

Man

tagon

ist3 x 10

-5M

+ kiss 10

-9M

antag

onist

5.6x 10

-5M

+ kiss 10

-9M

antag

onist

10-4

M+ kis

s 10-9

M

antag

onist

10- 4

M

kissp

eptin

10-9

M

Fold

incr

ease

inIP

accu

mul

atio

n


Neurodegeneration

IPIP--One ELISA assay of GPR54 with One ELISA assay of GPR54 with kisspeptinkisspeptin

-8 -7 -6 -5 -4 -30

50

100

1501e-8M hKISS1e-7M hKISS

Log [antagonist] (M)

B/B

0

Standard Curve - IP-One ELISA

-9 -8 -7 -6 -50

20

40

60

80

100

Log [IP1] (M)

B/B

0

Standard Curve - IP-One HTRF

-9 -8 -7 -6 -5 -40

500

1000

1500

Log [IP1] (M)

delta

F

ELISA is more sensitive then HTRF

Data very similar

Allows labs without fancy readers the ability to use IP-One


Neurodegeneration

• A GPR54 stably transfected cell line was generated for screening and was found to respond appropriately in functional assays.– IP accumulation– ERK phosphorylation

SUMMARYSUMMARYSUMMARY


Neurodegeneration



• An assay based on measurement of IP1 accumulation was optimized for high-throughput screening.


Neurodegeneration



• We completed an initial screen of an entire small molecule library for GPR54 agonists.



Neurodegeneration



• We completed an initial screen of an entire small molecule library for GPR54 agonists.– Positive hits from ligand screening were selected and

verified using the screening assay (IP-One) and secondary functional assays.

– The compound verified on secondary assays is now being tested in an animal model.



Neurodegeneration



• We completed an initial screen of an entire small molecule library for GPR54 agonists.– Positive hits from ligand screening were selected and

verified using the screening assay (IP-One) and secondary functional assays.

– The compound verified on secondary assays will then be tested in an animal model.


• Repeat screening of the library for GPR54 antagonists and enhancers is underway.


Neurodegeneration

Harvard Center for Neurodegeneration and Repair

Ross Stein, PhD, Director LDDN Marcie Glicksman, Sr.Director Leads DiscoveryGreg Cuny, Sr.Director Chemistry• Jake Ni• April Case• Mickey Huang In collaboration with:

Brigham and Women’s Hospital• Wendy Kuohung• Ursula Kaiser• Deepa MukhtyarMassachusetts General Hospital• William Crowley, Jr.• Stephanie Seminara• Samuel Posner

Laboratory for Drug Discovery in Neurodegeneration

with ip-one assay - cisbio · case study: hts of gpr-54 with ip-one assay marcie glicksman, phd...

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