Sanford-Burnham Medical Research Institute

(La Jolla, CA)

HTRF Symposium Avignon, France

April 26, 2013

HTRF®-powered detection of

ubiquitin-like protein signaling

and interactions

Eduard Sergienko, Ph.D.

Director, Assay Development

Topics

• SBMRI and CPCCG

• NIH Roadmap Initiative Screening Network

• UBL signaling cascades and related projects within CPCCG

• Acknowledgements

Infectious &

Inflammatory Disease

Center is established

Diabetes & Obesity

Research Center

established

Development of Sanford-Burnham

Medical Research Institute

1976 1981 1989 1999 2002 2004 2006 2007 2010 1996

Renamed:

Sanford-Burnham

M. R. Institute

National Cancer

Institute designation

conferred

La Jolla Cancer

Research Foundation

established

Renamed:

The Burnham

Institute

Del E. Webb Center for

Neurosciences and Aging

established

Sanford Center for

Children’s Health

Research established

4

Infectious &

Inflammatory Disease

Center is established

Diabetes & Obesity

Research Center

established

Development of Sanford-Burnham

Medical Research Institute

1976 1981 1989 1999 2002 2004 2006 2007 2010 1996

Renamed:

Sanford-Burnham

M. R. Institute

National Cancer

Institute designation

conferred

La Jolla Cancer

Research Foundation

established

Renamed:

The Burnham

Institute

Del E. Webb Center for

Neurosciences and Aging

established

Sanford Center for

Children’s Health

Research established

5

Orlando Est. 2007

La Jolla Est. 1976

•2 Sites of operation

•1200 People (over 500 Ph.D. scientists)

•89+ Faculty

•$175 MM annual operating budget

6

• Occupied: April 2009

• 175,000 sq ft facility

• Part of Lake Nona Medical City

• 14,000 sq ft AAALAC-certified vivarium (rats & mice)

• Largest GOLD LEED independent scientific facility in Florida

• ~30 Principal Investigators; ~300 employees (Phase I capacity)

Sanford-Burnham Medical Research Institute

(Florida site)

Sanford-Burnham’s Discovery Research Programs

Discovery

Research

Programs

NCI-Designated

Cancer

Center

Neuroscience,

Aging, & Stem

Cells

Tum

or

Mic

roe

nvi

ron

men

t

Sign

al T

ran

sdu

ctio

n

Ap

op

tosi

s &

Ce

ll D

eath

Res

ear

ch

Deg

ener

ativ

e D

ise

ase

Res

ear

ch

Stem

Cel

ls &

Reg

ener

ativ

e B

iolo

gy

Dev

elo

pm

ent

& A

gin

g

Infe

ctio

us

Dis

eas

es

Infl

amm

ato

ry D

ise

ase

s

Bio

info

rmat

ics

& S

yste

ms

Bio

logy

Met

abo

lic S

ign

alin

g &

Dis

eas

e

Car

dio

vasc

ula

r P

ath

ob

iolo

gy

RN

A B

iolo

gy

Gen

etic

Dis

eas

e

Tum

or

De

velo

pm

ent

Infectious &

Inflammatory

Diseases

Diabetes and

Obesity

Research

Sanford

Children’s Health

Research

Mu

scle

Dev

elo

pm

ent

& R

egen

erat

ion

7

Animal Resources

Animal Care & Procedures

In Vivo Analysis

Cardiometabolic Phenotyping

Model Organisms

Cell Imaging & Analysis

Histology and Molecular Pathology

Cell Imaging

Flow Cytometry

Genomics

DNA Analysis

Microarray & QPCR

HT DNA sequencing

Proteomics & Metabolomics

Proteomics

Metabolomics

Medicinal Chemistry & Pharmacology

Medicinal Chemistry

SAR by NMR

Experimental Pharmacology

Structural Biology

Crystallography

NMR

Protein Production & Analysis

Bioinformatics &

Data Management

Bioinformatics

Cheminformatics

3D Modeling (CADD)

High throughput screening

Assay Development

Libraries and HT Screening

Ultra-HTS

High Content Screening

Cheminformatics

Functional Genomics

siRNA/shRNA screening

Viral Vectors

Stem Cells

Embryonic Stem Cells

Electrophysiology

Shared Resources

Superior Technology Infrastructure

empowers world-class science

Drug Discovery - CPCCG

Conrad Prebys Center for Chemical Genomics

• Bicoastal operations (San Diego, CA and Orlando, FL)

• 1 of 4 comprehensive NIH MLPCN centers for the past 7 years

• 370K MLSMR library + 320K SBMRI collection; focused libraries

• Performed ~135 HTS-based projects, developed >300 secondary assays, generated >50 chemical probes

• Most of assays development and screened in 1536-well plates

S|B World Class Discovery Capabilities

>$30 MM capital investment

• 5 Fully Integrated Robotic Liquid Handling

Systems

(HRE unipod and tripod /1536 format), acoustic liquid dispensing, multiple plate readers, tip box & plate hotels, CO2 Incubators, etc.

• 4 Fully integrated HCS Systems – Robotics arms, tip wash & cell dispense, plate hotel

& incubator

– Live cell and 3D imaging

• Off-line Instruments & Workstation – 6 Plate readers, 2 Plate washers, 3 Bulk reagent

dispensers

– Plate Workstation: Plate sealer,

barcode labeler, 384/1536 liquid handler

– Liquid Handler Workstation 96/384/1536

– High Content Microscope

– GPCRs – 2 Hamamatsu FDSS-7000

Sanford-Burnham uHTS Facility (Orlando)

• Robot runs several screens simultaneously, permitting complex scheduling

• Swap out device “on the fly” & dock “off-line”

• Acoustic compound transfer (x2.5 nL) for 1° HTS & cherry-picking

• Perkin Elmer Opera HCS + Acapella software

Echo (5 total)

Bio RAPTR

and Combi

Dispensers

Carousels

(500 plates each)

Handoff

Press

Lid hotel

Incubator x 3

Opera

Waste V-spin

x2

Viewlux

FDSS Envision

MicroDocks

5-Echos w/ On-Board library

3-Arms (Staubli RX-160L)

5 Readers

~1 min/plate

1500 plate capacity

>1 MM data pts/day

Additional docks allow

Off-system devices to

Communicate w/Cellario™

scheduler & data acquisition modules

MLPCN

• Molecular Libraries Probe Production Center Network (MLPCN)

• Molecular Libraries Small Molecule Repository (MLSMR) maintains a collection that is shared by all centers in the network

• HTS and SAR data released into PubChem database Counter screen information

• Generate knowledge relevant to human health Target validation

Tool compounds for untargeted proteins and classes

Starting points for drug discovery

Specifics of MLPCN projects

MLPCN Center

PIs

PubChem

MLPCN

R03/X01 Outreach

Assignments & Funds

12-18 mos

• MLPCN centers funded by NIH Common Fund

• PIs access the network through MLPCN grants; the applications require working primary assays

• Part of Center’s budget allocated to outreach

The next page in academic screening

• A novel mechanism for funding the academic screening is currently being tested with PAR-12-058 (HTS R01), -059 (HTS R21) and -060 (MedChem R01)

• Grants require working assays and testing funnels

Screening Center

PIs

PubChem BARD

NIH IC

R01/R21

At-risk support

24-36 mos Funds

Funds

NIH Review

MLPCN screening at CPCCG

2005-2013

125 Library Screens

52 Probes Identified

63 (50%)

Biochemical

Screens

(22 probes)

48 (38%)

Cell-based

Pathway

Screens

(19 probes)

14 (12%)

High

Content

Screens

(11 probes)

Detection Format

Absorbance AlphaScreenFluorescence Fluorescence PolarizationFRET / TR-FRET High Content ScreenLuminescence NMR

Target Class

Cell Death Cell ProliferationEndonuclease GlycosylationGPCR IsomeraseKinase LigasePhenotypic Pathway PhosphataseProtease Protein LocalizationProtein-Nucleic Acid Interaction Protein-Protein InteractionReporter Gene ThioesteraseTransferase

UBL Cycle

GlyGly UBL

GlyGly O

O- UBL

ATP

PPi

E1

GlyGly O

S UBL

E1

GlyGly O

AMP UBL

Cys-SH

E1 Cys-SH

E2

HS-Cys

E2

S Lys-NH2

GlyGly O

S UBL

S

GlyGly O

NH UBL

Lys

Biological

effects

E3 DUB/ SENP

“Activation”

“Conjugation”

“Ligation”

MLPCN UBL projects performed at CPCCG

Projects PIs Polyubiquitination Dr. John Reed (SBMRI, La Jolla, CA)

SUMOylation Dr. Yuan Chen (City of Hope, Duarte, CA)

Neddylation Dr. Matt Petroski (SBMRI, La Jolla, CA)

SUMO-PPI Dr. Yuan Chen (City of Hope, Duarte, CA)

Senp1/CFP-SUMO1-YFP Dr. Jorge A. Iniguez-Lluhi (U. Michigan, Ann Arbor, MI)

Senp6/Z-RLRGG-AL Dr. Guy Salvesen (SBMRI, La Jolla, CA)

Senp7/Z-RLRGG-AL Dr. Guy Salvesen (SBMRI, La Jolla, CA)

Senp8/Z-RLRGG-AL Dr. Guy Salvesen (SBMRI, La Jolla, CA)

Senp8/Nedd8-AMC Dr. Guy Salvesen (SBMRI, La Jolla, CA)

Rpn11 Dr. Ray Deschaines (CalTech, Los Angeles, CA)

Csn5 Dr. Ray Deschaines (CalTech, Los Angeles, CA)

√

√

√

SUMO and human health

E1, E2, E3SUMO Protein-1SUMO

SIM

SUMO

Protein-1

Protein-2

isopeptidase

Breast, lung and skin cancers Metastasis

hypoxia

Most SUMO-dependent functions

~5-10% of genome

P53

Histone

HDAC

Cyclins

…

Viral proteins

HCMV IE1 &IE2

HIV P6-Gag

…

Prostate Cancer, Angiogenesis

SUMO PPI project

• PI: Yuan Chen, PhD (City of Hope, Duarte, CA)

SUMO1-PIAS2 peptide complex Song J, Zhang Z, Hu W, Chen Y, J Biol Chem (2005) 280: 40122

Protein -2

SUMO

Protein-1

SIM

SUMO PPI project

• PI: Yuan Chen, PhD (City of Hope, Duarte, CA)

• MLPCN grant R21 NS066498-01

Song et al, PNAS, 2004

Song et al, JBC, 2005

Assay design for SUMO PPI project

Biological reagents:

• SUMO1-GST

• SUMO2-GST

• SUMO3-GST

• FITC-S1 peptide (F-S1)

• S1-FITC peptide (S1-F)

• FITC-S2 peptide (F-S2)

Detection reagents:

• Lumi4® Tb anti-GST (CisBio; cat# 61GSTTLA)

Tested a matrix of two SIM peptides and three SUMO paralogues in three assay formats

Established two potential pairs for further assay development: SUMO1/S1 and SUMO2/S1

Development and validation of HTS assay

• Binding assay for GST-SUMO1/FITC-S1 pair developed with HTRF Lumi4®-Tb anti-GST antibodies for primary HTS

• Developed counter screen assays • Pilot HTS was performed on three assays

in 1536-well plates using PHERAstars • The project submitted into MLPCN

0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

S 1 F H T R F

F S 1 H T R F

E C 5 0 = 8 .7 u M

E C 5 0 = 1 .4 u M

[F lu o re s c e in -P e p tid e ] (n M )

TR

-FR

ET

ra

tio

(5

20

/49

0)

x 1

0,0

00

1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2

4

6S 1 -F H T R F

F -S 1 H T R F

F L U O R E S C E IN (n M )

S/B

G S T -ta g C H E C K k it (C is B io )

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

0 .1 1 1 0 1 0 0 1 0 0 0

G S T c o n tro l

G S T -S U M O 1

G S T -S U M O 2

[G S T d is p la c e r ] (n M )

TR

-FR

ET

(6

65

/62

0)

x1

00

00

0 20 400

5000

10000

15000

20000

GST-SUMO1/FITC-S1

FITC-S1 (no SUMO1)

IC50 = 11.9 uM

[S1 peptide] (uM)

FR

ET

RA

TIO

(5

20

/49

0)X

10

00

0

SUMO-PPI MLPCN screening project

• Primary HTS (PubChem AID 602429) • TR-FRET assay using GST-SUMO1 and FITC-S1 peptide and Lumi4®-

Tb anti-GST (1536-well plates)

• 364K-compound MLSMR collection

• Hits profiled against PubChem data to eliminate artifacts

• Secondary assays: • FP assay SUMO1 and FITC-S1 (PubChem AID 624382)

• TR-FRET SUMO2 and FITC-S1 (PubChem AID 624384)

• FP assay SUMO2 and FITC-S1 (PubChem AID 624385)

• PubChem Summary AID 602467

• PI lab is currently characterizing the compounds identified in the screening

Danielle Key, Ada Kane, Siobhan Malany, PhD

Eliot Sugarman, Kevin Nguyen, Eigo Suyama, PhD, Steve Vasile, PhD

Breast, lung

& skin cancers

Metastasis

SIM

SUMO

Protein-1

Protein-2

Most SUMO-dependent

functions

~5-10% of genomep53 Histone

HDAC Cyclins …

Viral proteinsHCMV IE1 & IE2

HIV P6-Gag …

E1, E2, E3SUMO

Protein-1SUMO

isopeptidase

Hypoxia Prostate Cancer, Angiogenesis

SUMOylation project

• PI: Yuan Chen, PhD (City of Hope)

• MLPCN screening grant R03 MH084862-01

SUMOylation project: HTRF Assay

Enzymes and substrates (PI lab) • E1 and E2 • GST-SUMO and His6-RanGAP1

Detection reagents (CisBio) • Anti-GST-XL665 (61GSTXLB)

• Anti-6HIS-Eu3+Cryptate (61HISKLB)

GST SUMO E1

ATP AMP

GST SUMO

E1 Cys

S

O

E2

RanGAP1 His6

E2 Cys

S

O

GST SUMO

GST SUMO NH

O

RanGAP1 His6

Anti-GST-XL665

Anti-6HIS-Eu3+Cryptate

GST SUMO NH

O

RanGAP1 His6

XL665

XL665

Ex340 Em665

FRET

SUMOylation project: HTS

• Primary HTS (PubChem AID 2006)

• 291K-compound MLSMR collection

• Hits profiled against PubChem data

• Secondary assays:

• HTRF interference assay (PubChem AID 2069)

• ALPHAscreen SUMOylation (PubChem AID 2018)

• TR-FRET Ubiquitination (PubChem AID 2658)

• PubChem Summary AID 2011

Sharon Colayco, Ekaterina Bobkova, PhD

Justin Rascon, Carlton Gasior, Fu-Yue Zeng, PhD

SUMOylation project: Probe compound

• The probe compound kills cancer cells

• The probe specifically inhibits SAE in the cells

Baozong Li, PhD, Yi-Jia Li, PhD, Yuan Chen, PhD

CUL1 Neddylation Project

• PI: Matt Petroski (SBMRI)

• The assay targeting NAE and Ubc12 designed and developed in collaboration with PI

Assay captures CUL1 neddylation

• Assay Reagents:

• NAE (His6-tag) and Ubc12 (His6-tag)

• SCF (Skp1-CUL1-F-box) – CUL1 is FLAG-tagged

• Nedd8 Fluorescein-Nedd8

Biotin-Nedd8

• Anti-FLAG antibodies Eu-anti-FLAG (Life Tech)

Lumi4®-Tb anti-FLAG (CisBio)

Streptavidin DyLight650 (Pierce; cat #84547)

Streptavidin DyLight488 (Pierce; cat #21832)

Ubc12

FITC Nedd8 NH

O

CUL1 (SCF) FLAG

FITC Nedd8 O

OH

NH2 CUL1 (SCF) FLAG Lys

ATP

AMP

NAE

Nedd8 and CUL1 reagents

• Biotin-Nedd8 assay demonstrated low S/B and required end-point assay mode

• FITC-Nedd8 is consistent with kinetic mode • Lumi4®-Tb anti-FLAG Ab has no effect on the reaction

• CUL1 with N-terminal FLAG tag provides higher S/B in the assay than C-terminal FLAG tag

0 50 100 150 2000

500

1000

1500

FLAG-SCF

SCF-FLAG

[SCF] (nM)

Flu

ore

sc

en

ce

Ra

tio

(5

20

/49

0)x

10

00

0

CUL1 neddylation assay development

• All assay parameters were optimized

• Many reagents tested in “matrix” experiments

0 5 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

6 0 m in

1 5 m in

3 0 m in

[ f lu o -N e d d 8 ] (n M )

Ra

tio

(5

20

/49

0)x

10

00

0

0 5 0 0 1 0 0 0

0

2

4

6

1 5 m in

3 0 m in

6 0 m in

[ f lu o -N e d d 8 ] (n M )

S/B

0 5 0 1 0 0 1 5 0 2 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

1 5 m in

3 0 m in

5 0 m in

[S C F ] (n M )

Ra

tio

(5

20

/49

0)x

10

00

0

0 50 100 150 2000

2

4

6

15 min

30 min

50 min

[SCF] (nM)

S/B

0 1 0 2 0 3 0 4 0 5 0

0

3 0 0 0

6 0 0 0

9 0 0 0

2

1

0 .5

0 .2 5

0 .1 2 5

0 .0 6 2 5

[A T P ] (u M )

L u m i4 -T b A b

0 10 20 30 40 500

1000

2000

3000

4000

0 nM NAE1

12.5 nM NAE1

50 nM NAE1

200 nM NAE1

Time (min)

Flu

ore

sc

en

ce

Ra

tio

(5

20

/49

0)

x1

00

00

CUL1 neddylation assay modalities

012.5

50200

0

500

1000

1500

2000

2500

3000

3500

4000

0

12.5

50

200

0

5 0

1 0 0

1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0

E 1 /E 2 p ro to c o l (5 0 u M )

E 2 p ro to c o l (2 0 0 u M A T P )

E 2 p ro to c o l (5 m M A T P )

[M L N 4 9 2 4 ] (n M )

Inh

ibit

ion

(%

)

• Fine-tuned enzyme and substrate concentrations and the order of reagent addition to establish two assay modalities (sensitive to inhibition of either E1&E2 or E2)

• Submitted to MLPCN (R03 grant DA034599-01)

CUL1 neddylation project summary

• 364K compounds screened in 1536-well plates (PubChem AID 651699)

• Hits profiled vs other HTS data

• 27 hit reconfirmed in dose response mode (PubChem AID 652247)

• All compounds appear to inhibit NAE

CV (positive control) 4.60%

CV (negative control) 6.50%

S/B 3.3 + 0.4

S/N 36.2 + 6.7

Signal window 13.1 + 3.6

Z' 0.75 + 0.04

hits* 2123

ordered hits 1922

% inhibition

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250 300

Z' f

acto

r

Plate Number

Probe compound is active in the cells

One NAE probe scaffold is active in cell-based cullin neddylation assay

MLN4924 has a complex mechanism of action

Brownell et al, 2010,

Mol cell 37:102-111

Overcoming MLN4924 resistance

• PI’s lab demonstrated that cancer cells could become resistant to MLN4924 through a single mutation in ATP or Nedd8 binding sites (Cell Rep. 2012, 1(4):309-16)

• The probe compound identified in our project kills MLN4924-resistant cells

MLN4924 SBI-620

Concluding remarks

• HTRF approach worked well for projects involving UBL signaling and interactions performed at CPCCG

• Several first-in-class chemical probes with biological activities in SUMOylation or neddylation systems were identified with the help of these assays

• Access to HTS data of diverse assays, e.g. with similar targets or similar detection, allows early identification of assay artifacts and promiscuous compounds

• Utilization of a reagent toolbox for diverse detection approaches helps with rapid hit validation and profiling

Acknowledgements

• Chen-Ting Ma

• Sharon Colayco

• Hung Nguyen

• Siobhan Malany

• Sylvia Kim

• Carlton Gasior

• Fu-Yue Zeng

• Thomas Chung

La Jolla, San Diego, CA

Lake Nona, Orlando, FL

• Yuan Chen (City of Hope)

• Larry Tong (COH)

• Aileen Alontaga (COH)

• Matt Petroski (SBMRI)

• Julia Toth (SBMRI)

• Michael Jackson

• Kristiina Vuori

• John Reed

NIH Roadmap grants:

5U54HG005033

5U54HG003916

R03 MH084862

R21 NS066498

R03 DA034599