Modifying the Traditional HTS Workflow

Sam Michael NIH Center for Translational Therapeutics Chicago, September 2011

NIH Center for Translational Therapeutics

• • • Founded 2004 as part of NIH Roadmap Molecular Libraries Initiative MLPCN (screening & chemical synthesis; compound repository; PubChem database; funding for assay, library and technology development ) • Complements individual investigator-initiated research programs • Enables “pharma-level” HTS and early chemical optimization – Develop new chemical probes for basic research and leads for therapeutic development, particularly for rare/neglected diseases – New paradigms & applications of HTS for chemical biology / chemical genomics Proposed component of NCATS ca. Oct 2011

Leveraging quantitative HTS (qHTS)

A Assay concentration ranges over 4 logs (high:~ 60

μ

M) 1536-well plates, inter-plate dilution series Assay volumes 5

μL

C Automated curve fitting and classification D B Automated concentration-response data collection

Biochemical assay Cell-based assay Simple design Complex design

Generic Assay Protocol

B000000n H000000n 1. Add Reagent to Assay Plate 2. Transfer Compound to Assay Plate 3. Add Detection Reagent to Assay Plate 4. Read Assay Plate 5. Dispose of Assay Plate

HTS Scheduling Methodology

• • • • Assay plates act as the input to our HTS system, and may have associated control and compound plates as required Each assay plate is considered to be an assay object A method is the set of steps each assay object will run, with each step usually associated with some peripheral device (dispenser, plate reader, etc.) A group of assay objects running the same method is considered to be an Assay

Wait Queue H0000002 H0000001 Mutex

Wait Queue and Mutex

Mutex • • • • • At the start of an Assay, all assay objects are placed in a first in, first out (FIFO) Wait Queue A Wait Queue is a queue of objects waiting for a lock, which in this case represents control of a peripheral device on the screening system When the lock is unlocked, the objects acquire the lock in the order of the queue Objects still in the queue are in the blocked state until the lock is released Each step or a series of linked steps in a method has an associated lock, with this type of synchronization being called a Mutex (mutual exclusion)

Traditional HTS Workflow

Assay Series 1 H0000011 H0000012 H0000013 Compound Series 1 B0000011 B0000012 B0000013 Assay Series 2 H0000021 H0000022 H0000023 Compound Series 2 B0000021 B0000022 B0000023 Assay Series 3 H0000031 H0000032 H0000033 Compound Series 3 B0000031 B0000032 B0000033 Assay Series N H00000N1 H00000N2 H00000N3 Compound Series N B00000N1 B00000N2 B00000N3 H000001n B000001n H000002n B000002n H000003n B000003n H00000Nn B00000Nn • • • • System setup (loading of plates, reagent addition, configuration of detectors, etc.) Run assay protocol for each plate, with a 1:1 relationship between assay plates to compound plates Analyze data upon completion Perform follow up or secondary assays

HTS System Monitoring and Control

• • • • Most HTS systems provide the capability to actively monitor events in real time and to control the provided scheduling software through external applications Some HTS systems allow multiple processes to run in parallel Some HTS systems allow the programmatic launch of new processes If these features are present, custom software can be developed to greatly enhance traditional HTS workflow models through event triggering based on some condition(s) being met

Assay Series N H00000N1 H00000N2 H00000N3 H00000Nn

Input Assay Plates Controlling Application

Monitoring Technique

Informatics

• Data Files

Output Data Files

• • For our system, the scheduling software provides an event that can be captured for every protocol step for every assay object currently running, giving a complete real time history An external application written in LabView is used to communicate with the scheduler to capture these events, which are stored in a buffer to be used to take some action programmatically This LabView application is also configured to communicate with other external applications to both send and receive data to and from

HTS Screening System

Event Types

• • • Per Plate: – For every plate that is actively running on the screening system, each method step is continuously monitored and stored. Since we can monitor every event for every plate, we can setup the transition from one method step to another to act as a trigger to perform some action. This is called the ‘Transition Step Index’.

Per Group: – Since we keep a data buffer of every plate on the system, groups of plates can be monitored for some condition to be met to trigger some action being taken.

External: – Since our application is configured to receive messages from external applications, actions can be triggered by the receipt of a message.

Action Examples

• Log to database • http://ncgcweb.nhgri.nih.gov/Kalypsys/index.php

• Run QC • Email alarm message • Initiate new assay • Send message to external application • Selective dispense

Buffered Biochemical Assay Protocol

B000000n H000000n 1. Add Reagent to Assay Plate 2. Transfer Compound to Assay Plate 3. Add Detection Reagent to Assay Plate 4. Read Assay Plate 5. Clean Assay Plate

Wash effectiveness:

Absorbance

Goal: determine the effectiveness of the wash protocol by dispensing concentrated bovine serum albumin (BSA) on 10 plates and testing for residual protein following washes Results: Protocol: 1.Dispense 5uL 1% BSA / well 2.Incubate for 30 min 3.Aspirate/wash protocol 4.Dispense Bradford protein detection reagent 5.Incubate 5 min and read Absorbance on ViewLux

NO DETECTABLE PROTEIN REMAINING

*Note: 1% BSA is 10x the normal assay concentration (0.1%), so this test represents a worst-case scenario for the washer

Wash effectiveness: Fluorescence

Goal: determine the effectiveness of the wash protocol by dispensing concentrated bovine serum albumin (BSA) on 10 plates and testing for residual protein following washes Protocol: 1.Dispense 5uL 1% BSA / well 2.Incubate for 30 min 3.Aspirate/wash protocol 4.Dispense Quant-It fluorescent protein detection reagent 5.Incubate 5 min and read fluorescence on ViewLux Results: •

Less than 1.5% BSA of original fluorescent signal remaining

•

Washer consistently removed 99.5+% of initial BSA

*Note: 1% BSA is 10x the normal assay concentration (0.1%), so this test represents a worst-case scenario for the washer

Effectiveness of compound removal

Wash test: • TANGO cell line: transgenic GPCR cell line • Generates FLuc expression in presence of agonist • Sensitive to low nM agonist concentrations 1.

2.

3.

4.

5.

6.

7.

Protocol: Plate TANGO cells in new plates Dose with LOPAC 1280 library (containing dozens of known B2AR agonists) Incubate overnight Add detection reagent and read WASH plates (2x EtOH, 2x diH 2 O) Rerun TANGO assay using washed plates, but without dosing Observe a) viability of cells and b) carryover of compound

LOPAC library response:

46uM 15uM

LOPAC library results

3uM DMSO

Same plates after washing and reseeding:

Agonist titration before/after wash

*no appreciable signal detected in washed plates (even in wells previously dosed with as much as 15uM agonist)

Determine Assay Plate Buffer Size

Step

1 2a, 2b 3 4a, 4b 5 6 7 8 9 Rate Limiting Step (9 min) RGS4 Online Collection 1536-well Protocol

Parameter

Reagent Control Cpd Time Reagent, Reagent Time Detector Time Detector Wash

Value

2 µL 23 nL, 23 nL 10 min 2 µL, 2 µL 15 sec, 1000 rpm 620 nm/688 nm (FP, S, and P) 90 min 620 nm/688 nm (FP, S, and P) 0 µL Cycle Time (90 minutes) How many assay plates maximum are active on the screening system at any one time?

(Assay duration) / (Rate of plate addition) = Number of assay plates on the system

This is one series of plates. If we add an additional series, we can create a Circular Buffer of plates to run an assay independent of the number of compound plates required. Two series of plates represents one Cycle.

Series Size = (90 min / 9 min) + 1 = 11

plates

Transition Step Index

Circular Buffer

Head (Extract) Assay Plate Series 2 Plates 12:22 Assay Plate Series 1 Plates 1:11

18 19 20 21 22 17 16 15 14 13 12 1 2 11 10 3 9 4 8 5 6 7

Tail (Insert) • The total size of the Circular Buffer is double the number of plates in one series, with this case being 22 total plates.

• At the start of a Buffered Assay, the first series of plates is extracted from the head of the buffer and launched as an assay, with the second series being inserted into the tail.

• For each plate in the series, once that plate is complete it is placed back into the buffer. Upon a determined trigger step for the last plate in a series, the next series of plates is extracted from the buffer and launched as a new assay.

• This process continues until all of the compounds for an assay have been screened.

Buffered Assay HTS Workflow

Assay Series 1 H0000011 H0000012 H0000013 H000001n Assay Series 2 H0000021 H0000022 H0000023 H000002n Compound Series 1 B0000011 B0000012 B0000013 Compound Series 2 B0000021 B0000022 B0000023 Compound Series 3 B0000031 B0000032 B0000033 Compound Series N B00000N1 B00000N2 B00000N3 B000001n B000002n B000003n B00000Nn • • • System setup (loading of plates, reagent addition, configuration of detectors, etc.) Run assay protocol for each plate, with a 1:N relationship between assay plates to compound plates Analyze data upon completion

Cycle 1

Trigger Step Index

Buffered Assay Example

Series 1 Series 2 • • • • Two parallel buffered assays running simultaneously 22 plates per assay with 11 plates per series, so a total of 44 plates used to screen against over 1000 compound plates Nearly 7 days continuous system runtime Other system resources still available during the course of the assays

Buffered Assay Benefits

• • • • • • Greatly reduces the initial start up time required to start a large assay Removes need for running large assays in a batch mode with interruption Reduces real estate requirement of loading hundreds of plates, freeing the system to run additional assays in parallel ‘Eco-Friendly’; Greatly reducing the plastic waste generated by the system

The typical Buffered Assay uses ~40 assay plates to screen ~1,500 compound plates Since March 2011, NCTT has used this technique to screen ~15,000 compound plates with ~750 assay plates, representing a cost savings of ~$140,000 in plates alone

1536-well W2C system

:

Multistage qHTS with a real time informatics platform

Robotics & Informatics Microliter Dispensing

1 1 2 2

Microscopy Laser Cytometry

Merging Screening Technologies

High throughput screening

• • • • Lead identification Single (few) read outs High-throughput Moderate data volumes • • • •

High content screening

Phenotypic profiling Multiple parameters Moderate throughput Very large data volumes • • We’d like to combine the technologies, to obtain rich high-resolution data at high speed Is this feasible? What are the trade-offs?

Merging Screening Technologies

• • A simple solution is to run a HTS & HCS as separate, primary & secondary screens Alternatively – Wells to Cells – Integrate HTS & HCS in a single screen using a combined platform for robotics & real time automated HTS analytics –

Selective imaging of interesting wells

Wells to Cells Workflow

• • • • • • Sequential qHTS using laser scanning cytometry followed by high-res microscopy Unit of work is a plate series The same aliquot is analyzed by both techniques A message based system – Persistent, can be kept for process tracking, reporting – Asynchronous, allows individual components of the workflow to proceed at their own pace – Modular, new components can be introduced at any time without redesigning the whole workflow We employ Oracle AQ, but any message queue can be employed

The key is deciding which wells go through the workflow

Induced excess DNA replication (EDR) in cancer cells

• • • Goal: identify small molecules that selectively induce DNA re-replication in cancer cells but not normal cells – (i.e. mimic the effect of geminin loss of function) There is no small molecules prior art Approach: use an image-based assay to measure DNA re-replication – Screen a tumor cell line (SW480) and a normal cell line (MCF10A) in parallel to identify tumor selective actives – Dynamically analyze series data generated by an Acumen eX3 to select wells to be screened by an INCell 1000 within four hours

Well Selection Criteria

• • Generally, pre-determined (from validation assays) Selection criteria implemented as Java code – Easy to adapt for different assays – Currently only makes use of the titration curve parameters – Could easily involve • Chemical structure • Enrichments • Predictive models

DNA Re-Replication Automation Workflow

Compound Series to Screen?

Acumen HTS Assay

SW480 Assay H0000011 H0000012 H0000013 B0000011 B0000012 B0000013 H000001n Plate Series B000001n MCF10A Assay H0000011 H0000012 H0000013 B0000011 B0000012 B0000013 H000001n Plate Series B000001n Acumen 1 Acumen 2 qHTS DB • • • All three processes run in parallel The HTS screening system acts as a listener, launching a new INCell HCS assay when a message is received from the Informatics Platform In the event of no selections being made for a compound series, no INCell HCS assay is launched

Informatics Platform

Data Processing Curve Fitting

INCell HCS Assay

SW480 and MCF10A Plate Series Acumen Queue qHTS DB Acumen Queue Well Selections INCell Queue INCell

DNA Re-Replication Statistics

• • • Selection Criteria – CRC 1a or 2a – CRC 1b >40%Efficacy T read – – distribution 6 plates/ series 134 min median Selected wells – 20 (median) INCell Inter-Plate titration Acumen CRC

Stage Primary qHTS Primary HCS qHTS HCS qHTS Cpds

343,078 343,078 5,218

Image collection time reduced From ~11 hrs/plate series to ~10 min.

(median of 20 selections) % SMR*

100 100

Samples

2,058,468 2,058,468

GBdata

0.241

54,000

Details

6 concs.; 2-cell lines 1.5

31,308 266 40X objective, 9 fields

0.50% of the storage space that would be required for imaging 2.0 M samples

Cell Cycle Assay

• Cell cycle regulation is related to cancer • Cell cycle protocol has been well validated for Acumen and 384 well format (Kittler et al. 2008; Low et al. 2008) • Multiple phenotypic sub-populations, such as %G1, S, G2/M are identified from each well using a nuclear stain (Hoescht) and measuring the total intensity •Histogram representation for the distribution of the total fluorescent intensity (FLU) obtained for nuclei objects in a representative well (left) -Can obtain multiple phenotypes per well •G1 and G2/M CRCs and structures for known representative actives (right) •1 field of view/1 nuclear stain for Acumen imaging

Cell Cycle Assay Improvement

• • Primary Acumen HTS assay (1 st – G2/M populations phase protocol) nuclear stain (DNA content) used to define the %G1, S, Select “active wells” from the Acumen HTS assay – User-defined thresholds based on efficacy, curve class, population of interest, etc and launch 2 nd phase of protocol – Utilize antibodies for better sense of mechanism of action – Launch an immunofluoresence assay using the same plates for “selective” well dispensing of precious reagent • Anti-Phosphohistone H3 which measure mitotic population • Anti  tubulin, which measures tubulin integrity/intensity

Step

1 (Day 1) 2 (Day 2) 3 (Day 3) 4 (Day 3) 5 (Day 3) 6 (Day 3) 7 (Day 3) 8 (Day 3) 9 (Day 3) 10 (Day 3)

Step

1 2 3 4 5 6 7 8 9 10

Parameter

Cells Drug Fixing PBS Washes PBS with Stain; Detector Analysis Blocking Antibody Stain PBS Washes Wash/Stain Detector

Value

6 µl 23 nl 4 µl 4 µl 4 µl 4 µl 4 µl 4 µl 6 µl Excitation/Emission

Description

Hela Controls/Compounds PFA PBS PBS/Hoechst; Acumen Read Blocking Buffer Antibodies PBS PBS/Hoechst In Cell

Notes

Plate HeLa cells with FRD (Aurora 1536 lobase clear bottom) and incubate in 37 ° C.

Pin after ~24-30 hours post plating. Return to Incubator for 24 hours.

Aspirate media with Kalypsys dispenser and add 4 µl PFA to wells for 20 min RT. Aspirate PFA with Kalypsys dispenser and add 4 µl PBS with Triton-X.

Aspirate PBS. Add Hoechst to PBS wash and incubate at room temp (covered/dark) for 20 minutes. Read on Acumen (Excitation/Emission) filter.

On the fly Analysis for well picking/selective reagent dispensing Aspirate PBS and add 4 µl blocking buffer with Triton-X for 1 hour room temp.

Aspirate blocking buffer and add 4 µl blocking buffer with conjugated antibodies (PHH3-488 and Tubulin-533) and Triton-X. Leave covered/dark for 2 hours room temp.

Aspirate antibodies and wash with PBS+Triton-X.

Aspirate PBS and add PBS with Hoechst. Leave 20 min room temp (covered/dark).

Image 20X (multiple fields).

Cell Cycle Automation Workflow

Compound Series to Screen?

• • • All three processes run in parallel The HTS screening system acts as a listener, launching a new INCell HCS assay when a message is received from the Informatics Platform In the event of no selections being made for a compound series, no INCell HCS assay is launched

Acumen HTS Assay

Cell Cycle Assay H0000011 H0000012 H0000013 B0000011 B0000012 B0000013 H000001n Plate Series B000001n Acumen qHTS DB • • The Acumen HTS Assay has a ‘Per Group’ Trigger, sending a message to the Acumen Queue to initiate processing for each plate series The INCell HCS Assay has a ‘Per Plate’ Trigger, using the well coordinates to selectively dispense conjugated antibody for each plate in a series to only active wells Acumen Queue Plate Series Data Processing Curve Fitting

Informatics Platform

INCell Queue Well Selections

INCell HCS Assay

Selective Dispense INCell

Cell Cycle Results

• Cost of conjugated antibody from Cell Signaling Technology - ~$250/100uL (PHH3 Alexa 488 and Tubulin Alexa 555 conjugated) • • We used a 1:100 dilution, with the protocol calling for: - 4uL/well or around ~6.2mL/plate - Full plate costing ~$150 or ~$0.10/well - Assay cost prohibitive for a large compound library By limiting the well selection with the primary screen, selecting only 100 wells would cut the cost of the screen down to ~$10/plate while providing a richer data set C7 C1 Hoechst PHH3  tubulin

Trade-offs & Opportunities

• • Automation reduces the ability to handle unforeseen errors – Dispense errors and other plate problems – Well selection based on curve classes may need to be modified on the fly Well selection does not consider SAR – Wells are selected independently of each other – If we could model SAR on the fly (or from validation screens), we’d select multiple wells, to obtain positive and negative results

Conclusions

• • Modifications to the traditional HTS workflow can result in significant savings of time and money by greatly reducing the resources required of both personnel and consumables without sacrificing data integrity Straight forward monitoring and controlling techniques coupled with simple event handling can greatly enhance the types of assays that can be run on a HTS system

• • • • • • • • • Jim Inglese Sunita Shukla Raj Guhar Kyle Brimacombe Ronald Johnson Doug Auld Trung Nguyen Steve Titus Jennifer Wichterman

Acknowledgments