Lead ID Department: Capabilities

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Lead ID DepartmentCapabilities Collaboration
OpportunitiesPeter Hodder
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Presentation Overview

• Introduction to HTS Lead ID
• Lead ID Resources
• Mechanisms for collaboration

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Lead Identification by HTS Funneling
Methods(GPCR Agonist uHTS Example)
1MM Compound Library
2058
Primary Screen
Activity gt30 in 1º HTS Campaign (FLIPR)
658
Confirmation Screen
Confirm Activity gt30 in triplicate
252
Hit-Picking
Selected for Aequorin Assays
133
Secondary Screen
Activity gt40 _at_ 1mM Test Concentration in
Aequorin assay
Leads Identified
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IC50 in Aequorin Assay lt 10 mM
Screening techniques are effective at identifying
relevant therapeutic leads
Hodder et al., SBS, 2002
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Scale of HTS Assays
96 Well format (300 mL)
384 Well format (100 mL)
1536 Well format (10 mL)
Miniaturized 1536 wells/plate uHTS100,000
samples/24 hours
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Lead ID Charter

• Assist researchers with HTS assay development
• Consult on HTS-friendly formats
• Develop automate HTS/uHTS Assays
• Miniaturize assays to 1536 well format as
  appropriate
• Support Scripps HTS uHTS activities
• Manage Scripps HTS compound libraries
• Explore and implement novel screening
  technologies

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Lead ID Resources
Facilities Assay Development Lab uHTS
Automation Compound Management Automation
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TSRI uHTS Facility
1810 ft2 automation room, gowning chamber 310 ft2
assay implementation lab 80 kW UPS Environmentally
monitored controlled clean-room
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HTS Assay Development Lab
Reagent Cell Dispensing
Reagent Cell Dispensing
Tissue Culture Suite
Pipetting
Product Detection
Off-line 96/384/1536 wf Equipment
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uHTS Automation Platform
Compound Assay Plate Storage
Dispensers Aspirators
Stäubli RX130L robotic arm
Compound Transfer Station
Plate Reader
Supports Cell based biochemical assays Robust
high-quality data in 1536 wf
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Formats Amenable to uHTS Miniaturization

• Absorbance
• Melanophores (various receptors)
• Fluorescence intensity
• Beta-lactamase reporter gene (GPCRs, NHRs, etc.)
• Fluorescence Dequenching (Proteases)
• FRET (kinases)
• QTL (kinases)
• Fluorescence Polarization
• Kinases (IMAP)
• Luminescence
• Luciferase reporter gene (various receptors)
• Cell-titer Glo (Viability/Apoptosis)
• EFC (cell-based cAMP assays)
• Time-resolved fluorescence
• HTRF (Kinase Assays, cell-based cAMP
  measurements)
• DELFIA (ELISA assays)

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Lead ID Resources CM Automation
Cherry Picking to 384 1536 well plates
Temperature gas-controlled storage of 746K
samples in 384 well format
Customized Software Automation
Rapid, automated access to any compound library
member
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TSRIs HTS Compound Libraries

• LOPAC Tocris
• 2,200 compounds
• All compounds have known pharmacologic activity
• Useful for target identification/validation
• Non-proprietary, no publishing restrictions
• TSRI
• 600,000 compounds, from commercial vendors
  in-house research efforts
• Knownish (e.g. Kinase-specific, GPCR-specific)
  unknown pharmacologic activity
• Use is exclusive to TSRI researchers
• MLSCN (NIH)
• 65,000 compounds
• Known unknown pharmacologic activity
• Data acquired from HTS effort uploaded to PubChem
• Accessed through NIH funding mechanism

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Setting up HTS Collaborations
Business Rules
How many HTS campaigns are scheduled? What are
the collaboration mechanisms? What is the
protocol for collaboration?
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Typical Miniaturized uHTS Campaign Workflow
Process
Deliverable
Assay Development
384 well Assay
1536 well Assay
HTS Assay Miniaturization
Preliminary HTS Data
Robotic Validation
Data Compound act or inh _at_ single
concentration (singletons)
Primary HTS Campaign
Data Hit List, max 5120 for 600K assay
Selection of Hits
Plates 5120 Compounds _at_ 1 mM, 7 uL/well in 1536
wf
Hit-Picker Campaign
Data Hit Reproducibility (triplicate), act or
inh
Confirmation Assay
Data Hit Selectivity (triplicate), act or inh
X Counterscreen Assays
Plates max 128 Titrated Compounds in 1536 wf for
600K assay, compoundo2.5 mM,10-point DRC
Hit-Picker Campaign
Data EC/IC50, hill slope, max, min
Titration Assays
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Scheduling a uHTS Campaign
HTS Assay Miniaturization
1º HTS Campaign
Hit Picking
Confirmation Assay
Data Publication
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Sources of HTS Campaigns
DD
MLSCN
Intramural
Florida
Ext. Collab.
Lead ID can perform 20 uHTS Campaigns in 2006,
and can assist researchers w/ offline assay
development
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Mechanisms for Collaboration(Available only to
TSRI Researchers)

• Shared Library
• Assay feasibility studies
• HTS assay development
• MLSCN library HTS effort
• TSRI library HTS effort

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Shared Library Collaboration (no screening
effort)
Process
Deliverable
Hit Pick List
Hit List for Cherry Pick Robot
SL Plate 320 Compounds _at_ 1 mM, 7 uL/well in 384
wf
Hit-Picker Campaign
Project Close
SL Plate, sd file Released to Project Scientist
Useful for exploratory research Justification
registration of request required One plate
created per faculty member Requests will be
refused if not enough compound exists
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Assay Feasibility Collaboration (LOPAC Tocris
Library Access)
Process
Deliverable
Assay Development
If feasible, a 96 or 384 well format Assay
Data Compound act or inh _at_ single
concentration, in triplicate
Lopac/Tocris Screen

Data Released to Faculty Member
Data assay protocol suitable for HTS assay
development or HTS campaign grant application
Ideal for generating preliminary assay data
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HTS Assay Development Collaboration
Process
Deliverable
384 well format Assay(s) suitable for HTS
validation
Assay(s) Development
Validated 384 well format assay
Lopac/Tocris Screen
Assay(s) Miniaturization
1536 well format assays suitable for HTS
validation
Lopac/Tocris Screen
Validated 1536 well format assay
Several variations possible- Can be customized
to the needs of the researcher Various funding
mechanisms available http//nihroadmap.nih.gov/gr
ants/index.asp, (R21/R03 funding mechanisms)
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MLSCN HTS Effort (NIH Compound Library)
Process
Deliverable
Assay Development
384 well Assay
Assay Miniaturization
1536 well Assay
Preliminary HTS Data
Robotic Validation
Data Compound act or inh _at_ single
concentration (singletons)
Primary HTS Campaign
Data Hit List (max 1280 compounds)
Selection of Hits
Plates 1280 Compounds _at_ 1 mM, 7 uL/well in 1536
wf
Hit-Picker Campaign
Data Hit Reproducibility (triplicate), act or
inh
Confirmation Assay
2nd Selection of Hits
Data Hit List (max 64 compounds)
Plates Top 64 Titrated Compounds in 1536 wf,
compoundo2.5 mM,10-point DRC
Hit-Picker Campaign
Data EC/IC50, hill slope, max, min Output
reviewed by Scripps Med Chem All Data to Pubchem
Titration Assays
Opportunity to discover chemical probes All
results published to PubChem
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TSRI Library HTS
Assay Development
384 well Assay
1536 well Assay
Assay Miniaturization
Preliminary HTS Data
Robotic Validation
Data Compound act or inh _at_ single
concentration (singletons)
Primary HTS Campaign
Data Hit List, max 5120 for 600K assay
Selection of Hits
Plates 5120 Compounds _at_ 1 mM, 7 uL/well in 1536
wf
Hit-Picker Campaign
Data Hit Reproducibility (triplicate), act or
inh
Confirmation Assay
Data Hit Selectivity (triplicate), act or inh
X Counterscreen Assays
Plates max 128 Titrated Compounds in 1536 wf for
600K assay, compoundo2.5 mM,10-point DRC
Hit-Picker Campaign
Data EC/IC50, hill slope, max, min
Titration Assays
Customized to the needs of the researcher- Any
amount of compounds can be screened against any
number of assays
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Protocols for Collaboration
Contact Lead ID to discuss goals of
research (schedule assay development
meeting) Determine appropriate collaboration
Mechanism (choose existing or custom
collaboration mechanism) Enter assay queue (All
official collaborations executed
first-come/first-serve) Schedule Assay Transfer
Meeting (when resources at Lead ID
available) Plan ahead for time sensitive
deadlines!
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Contact Information hodderp_at_scripps.edu (561)
799-8839