Sanford-Burnham

HTRF Symposium
Avignon, France
April 26, 2013
Sanford-Burnham
Medical Research Institute
(La Jolla, CA)

HTRF ® -powered detection of
ubiquitin-like protein signaling
and interactions
Eduard Sergienko, Ph.D.
Director, Assay Development

• SBMRI and CPCCG
• NIH Roadmap Initiative Screening
Network
• UBL signaling cascades and related
projects within CPCCG
• Acknowledgements
Topics

Development of Sanford-Burnham
Medical Research Institute
National Cancer
Institute designation
conferred
Renamed:
The Burnham
Institute
Infectious &
Inflammatory Disease
Center is established
Diabetes & Obesity
Research Center
established
1976 1981 1989 1996 1999 2002 2004 2006 2007 2010
La Jolla Cancer
Research Foundation
established
Del E. Webb Center for
Neurosciences and Aging
established
Sanford Center for
Children’s Health
Research established
Renamed:
Sanford-Burnham
M. R. Institute
4

Development of Sanford-Burnham
Medical Research Institute
National Cancer
Institute designation
conferred
Renamed:
The Burnham
Institute
Infectious &
Inflammatory Disease
Center is established
Diabetes & Obesity
Research Center
established
1976 1981 1989 1996 1999 2002 2004 2006 2007 2010
La Jolla Cancer
Research Foundation
established
Del E. Webb Center for
Neurosciences and Aging
established
Sanford Center for
Children’s Health
Research established
Renamed:
Sanford-Burnham
M. R. Institute
La Jolla
Est. 1976
•2 Sites of operation
•1200 People (over 500 Ph.D. scientists)
•89+ Faculty
•$175 MM annual operating budget
Orlando
Est. 2007
5

Sanford-Burnham Medical Research Institute
(Florida site)
• 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)
6

Sanford-Burnham’s Discovery Research Programs
Tumor Microenvironment
Tumor Development
Signal Transduction
Apoptosis & Cell Death Research
Degenerative Disease Research
Stem Cells & Regenerative Biology
Development & Aging
Infectious Diseases
Inflammatory Diseases
Bioinformatics & Systems Biology
Metabolic Signaling & Disease
Cardiovascular Pathobiology
RNA Biology
Genetic Disease
Muscle Development & Regeneration
Discovery
Research
Programs
NCI-Designated
Cancer
Center
Neuroscience,
Aging, & Stem
Cells
Infectious &
Inflammatory
Diseases
Diabetes and
Obesity
Research
Sanford
Children’s Health
Research
7

Superior Technology Infrastructure
empowers world-class science
Animal Resources
Animal Care & Procedures
In Vivo Analysis
Cardiometabolic Phenotyping
Genomics Model Organisms
DNA Analysis
Microarray & QPCR
HT DNA sequencing
Stem Cells
Embryonic Stem Cells
Electrophysiology
Functional Genomics
siRNA/shRNA screening
Viral Vectors
Proteomics &
Metabolomics
Proteomics
Metabolomics
Shared
Resources
Cell Imaging & Analysis
Histology and Molecular Pathology
Cell Imaging
Flow Cytometry
Bioinformatics &
Data Management
Bioinformatics
Cheminformatics
3D Modeling (CADD)
Drug Discovery - CPCCG
Structural Biology
Crystallography
NMR
Protein Production &
Analysis
Medicinal Chemistry &
Pharmacology
Medicinal Chemistry
SAR by NMR
Experimental Pharmacology
High throughput
screening
Assay Development
Libraries and HT Screening
Ultra-HTS
High Content Screening
Cheminformatics

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, CO 2 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
5-Echos w/ On-Board library
3-Arms (Staubli RX-160L)
5 Readers
Viewlux
V-spin
x2
Waste
Opera
Handoff
Press
Lid hotel
Incubator x 3
Additional docks allow
Off-system devices to
Communicate w/Cellario
scheduler & data acquisition modules
Echo (5 total)
Bio RAPTR
and Combi
Dispensers
FDSS
Envision
Carousels
(500 plates each)
MicroDocks
~1 min/plate
1500 plate capacity
>1 MM data pts/day

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 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
MLPCN
Assignments
& Funds
MLPCN
Center
R03/X01
Outreach
12-18 mos
PubChem
PIs

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
NIH
Review
R01/R21
Funds
Screening
Center
At-risk
support
24-36 mos
PubChem
BARD
NIH IC
Funds
PIs

MLPCN screening at CPCCG
Detection
Format
63 (50%)
Biochemical
Screens
(22 probes)
48 (38%)
Cell-based
Pathway
Screens
(19 probes)
14
(12%)
High
Content
Screens
(11 probes)
Target
Class
Absorbance
Fluorescence
FRET / TR-FRET
Luminescence
AlphaScreen
Fluorescence Polarization
High Content Screen
NMR
2005-2013
125 Library Screens
52 Probes Identified
Cell Death
Endonuclease
GPCR
Kinase
Phenotypic Pathway
Protease
Protein-Nucleic Acid Interaction
Reporter Gene
Transferase
Cell Proliferation
Glycosylation
Isomerase
Ligase
Phosphatase
Protein Localization
Protein-Protein Interaction
Thioesterase

UBL Cycle
UBL
GlyGly
ATP
PP i
UBL
O
GlyGly
AMP
Cys-SH
E1
O
UBL GlyGly
S E2
“Activation”
E1
E1 Cys-SH
HS-Cys
UBL
GlyGly
O
O -
“Conjugation”
DUB/
SENP
UBL
GlyGly
O
NH
Lys
E3
UBL
GlyGly
O
S
E2
Biological
effects
S
“Ligation”
S
Lys-NH2

MLPCN UBL projects performed at CPCCG
√
√
√
Projects
Polyubiquitination
SUMOylation
Neddylation
SUMO-PPI
Senp1/CFP-SUMO1-YFP
Senp6/Z-RLRGG-AL
Senp7/Z-RLRGG-AL
Senp8/Z-RLRGG-AL
Senp8/Nedd8-AMC
Rpn11
Csn5
PIs
Dr. John Reed (SBMRI, La Jolla, CA)
Dr. Yuan Chen (City of Hope, Duarte, CA)
Dr. Matt Petroski (SBMRI, La Jolla, CA)
Dr. Yuan Chen (City of Hope, Duarte, CA)
Dr. Jorge A. Iniguez-Lluhi (U. Michigan, Ann Arbor, MI)
Dr. Guy Salvesen (SBMRI, La Jolla, CA)
Dr. Guy Salvesen (SBMRI, La Jolla, CA)
Dr. Guy Salvesen (SBMRI, La Jolla, CA)
Dr. Guy Salvesen (SBMRI, La Jolla, CA)
Dr. Ray Deschaines (CalTech, Los Angeles, CA)
Dr. Ray Deschaines (CalTech, Los Angeles, CA)

SUMO and human health
Breast, lung and skin cancers
Metastasis
hypoxia
SUMO
E1, E2, E3
isopeptidase
Prostate Cancer, Angiogenesis
SUMO
SIM
Protein-1
Protein-2
Most SUMO-dependent functions
SUMO
Protein-1
~5-10% of genome
P53
Histone
HDAC
Cyclins
…
Viral proteins
HCMV IE1 &IE2
HIV P6-Gag
…

SUMO PPI project
• PI: Yuan Chen, PhD (City of Hope, Duarte, CA)
Protein-1
SUMO
SIM
Protein -2
SUMO1-PIAS2 peptide complex
Song J, Zhang Z, Hu W, Chen Y,
J Biol Chem (2005) 280: 40122

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

T R -F R E T (6 6 5 /6 2 0 ) x 1 0 0 0 0
T R -F R E T ra tio (5 2 0 /4 9 0 ) x 1 0 ,0 0 0
FRET RATIO (520/490)X10000
S /B
Development and validation of HTS assay
6 0 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
G S T -ta g C H E C K k it (C is B io )
G S T c o n tro l
G S T -S U M O 1
G S T -S U M O 2
4 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
6
4
S 1 -F H T R F
F -S 1 H T R F
2 0 0 0
1 0 0 0
2 0 0 0 0
2
0
0 .1 1 1 0 1 0 0 1 0 0 0
[G S T d is p la c e r] (n M )
0
0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0
[F lu o re s c e in -P e p tid e ] (n M )
• 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
20000
15000
10000
0
1 0 1 0 0 1 0 0 0 1 0 0 0 0
5000
F L U O R E S C E IN (n M )
GST-SUMO1/FITC-S1
FITC-S1 (no SUMO1)
IC 50 = 11.9 uM
0
0 20 40
[S1 peptide] (uM)

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

• PI: Yuan Chen, PhD (City of Hope)
SUMOylation project
• MLPCN screening grant R03 MH084862-01
Hypoxia
Breast, lung
& skin cancers
SUMO
E1, E2, E3
isopeptidase
Prostate Cancer, Angiogenesis
Metastasis
SUMO
~5-10% of genome
p53
HDAC
Protein-1
Histone
Cyclins …
Viral proteins
HCMV IE1 & IE2
HIV P6-Gag …
SUMO
SIM
Protein-1
Protein-2
Most SUMO-dependent
functions

SUMOylation project: HTRF Assay
GST SUMO
ATP
E1
AMP
GST SUMO
E1
O
S
Cys
E2
GST SUMO
E2
O
S
Cys
RanGAP1 His 6
GST SUMO
NH
O
GST SUMO
NH RanGAP1 His 6
Em665
XL665
FRET
O
RanGAP1 His 6
XL665
Ex340
Anti-GST-XL665
Anti-6HIS-Eu 3+ Cryptate
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)

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

• PI: Matt Petroski (SBMRI)
CUL1 Neddylation Project
• The assay targeting NAE and Ubc12 designed
and developed in collaboration with PI

• Assay Reagents:
Assay captures CUL1 neddylation
• 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)
FITC Nedd8 O NH 2 Lys CUL1 (SCF) FLAG
OH
ATP
AMP
FITC Nedd8
O
NH
NAE
Ubc12
CUL1 (SCF) FLAG

Fluorescence Ratio (520/490)x10000
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
1500
1000
FLAG-SCF
SCF-FLAG
500
0
0 50 100 150 200
[SCF] (nM)

R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0
Fluorescence Ratio (520/490) x10000
S /B
R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0
S/B
CUL1 neddylation assay development
6 0 0 0
6
6 0 0 0
6
4 0 0 0
4
4 0 0 0
4
1 5 m in
2 0 0 0
3 0 m in
6 0 m in
2
1 5 m in
3 0 m in
6 0 m in
2 0 0 0
1 5 m in
3 0 m in
5 0 m in
2
15 min
30 min
50 min
0
0 5 0 0 1 0 0 0
[flu o -N e d d 8 ] (n M )
0
0 5 0 0 1 0 0 0
[flu o -N e d d 8 ] (n M )
0
0 5 0 1 0 0 1 5 0 2 0 0
[S C F ] (n M )
0
0 50 100 150 200
[SCF] (nM)
4000
9 0 0 0
L u m i4 -T b A b
0 .0 6 2 5
3000
0 nM NAE1
12.5 nM NAE1
50 nM NAE1
200 nM NAE1
6 0 0 0
0 .1 2 5
0 .2 5
2000
0 .5
3 0 0 0
1
1000
2
0
0 10 20 30 40 50
Time (min)
• All assay parameters were optimized
0
0 1 0 2 0 3 0 4 0 5 0
[A T P ] (u M )
• Many reagents tested in “matrix” experiments

In h ib itio n (% )
CUL1 neddylation assay modalities
4000
3500
3000
2500
2000
1500
1000
500
0
200
50
12.5
0
0
200
50
12.5
1 0 0
5 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 )
1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
[M L N 4 9 2 4 ] (n M )
• 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)

Z' factor
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
1
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250 300
Plate Number
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

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
• Yuan Chen (City of Hope)
• Sharon Colayco
• Larry Tong (COH)
• Hung Nguyen
• Aileen Alontaga (COH)
• Siobhan Malany
• Matt Petroski (SBMRI)
• Sylvia Kim
• Julia Toth (SBMRI)
• Carlton Gasior
• Michael Jackson
• Fu-Yue Zeng
• Thomas Chung
• Kristiina Vuori
• John Reed
Lake Nona, Orlando, FL
La Jolla, San Diego, CA
NIH Roadmap grants:
5U54HG005033
5U54HG003916
R03 MH084862
R21 NS066498
R03 DA034599