SMALL MOLECULE SCREENS

1
A Workshop on High Throughput / High Content
Screening Applications to Target-based Drug
Discovery Research
SMALL MOLECULE SCREENS
Clifford Stephan, Ph.D. Research Assistant
Professor John S. Dunn GCC Chemical Genomics
Research Consortium
Scott R. Gilbertson, Ph.D. Professor M.D.
Anderson Chair in the College of Natural Sciences
and Mathematics Department of Chemistry,
University of Houston
2
Stages of Commercial Drug Development
Basic Research
Target Validation
HTS and Lead Id
Lead Opt
Preclinical Tox
File IND
Clinical Phase 0/1
Clinical Phase 2
Sales Marketing Phase 4
Clinical Phase 3
File NDA
Approval
Drug Development is a game of attrition. The
Challenge Select 1-2 compounds from the
millions of possibilities that will be safe and
efficacious in humans
3
Stages of Academic Drug Development
Basic Research
HTS and Lead Id
Lead Opt
Still a game of attrition. The Challenge
Identify agents that increase the fundamental
scientific knowledge for a particular target
with the possibility of providing further
validation of the target as a druggable
target. Retain the possibility of identifying a
lead series of compounds that could take our
research in new, unexpected directions. The
possibility of establishing intellectual property
and the basis for a future pharmaceutical.
4
Why perform High Throughput Screening?
HTS enables the testing of large numbers of
chemical substances for activity in diverse areas
of biology in a relatively short time. The
entire chemical space of small organic molecules
is estimated to be gt 1060. Of those, 2.7 x 107
compounds have been registered and made. (Nature
Insight, 2004) Responses studied can range from
biochemical systems of purified proteins or
enzymes to signal transduction pathways to
complex cellular networks (Systems Biology).
5
High Throughput Screening A relative term
HTS in Pharma and Biotech is a process loosely
defined as testing 10,000 to 100,000 data
points/day using industrialized methods
UltraHTS test gt1,000,000 data points/day
HTS in the Dunn Screening Core the potential of
screening 100s to gt10,000 data points/day
following Pharma industry standards
10,000 Compounds/day 100,000 Compounds/day
96-Well plate(80 compounds/plate) 125 Plates/day 1250 Plates/day
384-Well plate(320 compounds/plate) 32 Plates/day 313 Plates/day
1536-Well plate(1280 compounds/plate) 8 Plates/day 78 Plates/day
10,000 Compounds/day
96-Well plate(80 compounds/plate) 125 Plates/day
384-Well plate(320 compounds/plate) 32 Plates/day
1536-Well plate(1280 compounds/plate) 8 Plates/day
6
The Screening Continuum
From a poster by RR Tice et al of the National
Toxicology Program HTS Initiative, 2007
7
Classes of HTS Assays
Homogeneous Mix and Read style assays Simple
with addition steps only, higher
throughput Examples Cell viability Live
cell imaging Proximity (e.g., radioisotope,
FRET, ALPHA) Enzyme Kinetics Heterogeneous Tra
ditional style assays Multiple steps, more
manipulations, slower throughput Examples Trad
itional binding assays Traditional sandwich
ELISA
8
Compare Traditional Assays with HTS Assays
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Assay Volume 0.1 to 1 mL lt 1 µl to 100 µl
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Assay Volume 0.1 to 1 mL lt 1 µl to 100 µl
Reagents Varied Limited quantity Various batches Limited stability Consistent QS for complete screen Single batch - preferred Prolonged stability
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Assay Volume 0.1 to 1 mL lt 1 µl to 100 µl
Reagents Varied Limited quantity Various batches Limited stability Consistent QS for complete screen Single batch - preferred Prolonged stability
Variables Many Time/temperature Ligand/substrate concentration Cell type Compound
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Assay Volume 0.1 to 1 mL lt 1 µl to 100 µl
Reagents Varied Limited quantity Various batches Limited stability Consistent QS for complete screen Single batch - preferred Prolonged stability
Variables Many Time/temperature Ligand/substrate concentration Cell type Compound
Readout Time Milliseconds to months Minutes to hours
Parameter Traditional Assay HTS Assay
Protocol Complex, numerous steps Multiple Additions Multiple Aspirations Multiple Washes Simple, few steps (3-5) Additions Aspirations - discouraged Washes - discouraged
Assay Container Varied Tubes/slides/dishes/cuvettes/animals Microtiter plates 96/384/1536-Wells
Assay Volume 0.1 to 1 mL lt 1 µl to 100 µl
Reagents Varied Limited quantity Various batches Limited stability Consistent QS for complete screen Single batch - preferred Prolonged stability
Variables Many Time/temperature Ligand/substrate concentration Cell type Compound
Readout Time Milliseconds to months Minutes to hours
Output Formats Varied Readers/scoring/image interpretation Plate reader Absorbance/fluorescence/luminescence
Parameter Traditional Assay HTS Assay
9
Key factors for successful HTS

• time/well
• wells/day
• screens/year
• project time

Time
HTS Screen
Costs
Quality

• reagents
• consumables
• instrumentation
• personnel

• few false positives
• few false negatives
• SN,SW,z-Factor
• Validated Hits

10
HTS An Iterative Process
HTS Group Perform Primary Screen Purpose
Identify a starting place Method Interrogate
libraries of compounds/genes
Research groups Target Id and Validation Develop
Primary and Secondary assays Define criteria for
active compounds Direct Hit improvement process
HTS Group Secondary Screen Purpose Validate
initial Hits Method Selection of compounds or
medicinal chemistry
Chemistry groups Analysis and interpretation of
Data for Structure Activity Relationships Refine
and improve identified Hits Modeling and
medicinal chemistry Selection of compounds for
screening via virtual screening, focused libraries
11
Critical Issues to be Addressed Prior to Testing
the First Compound

• Key factors that must be addressed prior to
  screening
• Assay protocol (miniaturization/simplification)
• DMSO resistance (test 0.1 - 5), standard
  compound vehicle
• Reagent quantity and batch consistency
• Reagent stability for storage and use under
  assay conditions
• Appropriate positive and negative controls
• Assay reproducibility and signal stability
• Available secondary or counter screen to test
  target
• specificity and selectivity

12
Consider Reagent Quantity Compare traditional
assays with HTS assays
Traditional Assay 96-Well HTS 384-Well HTS
Plates/Day 1 40 40
Total Plates 5 375 94
Cells/Day 1 x 106 40 x 106 40 x 106
Total Cells 5 x 106 375 x 106 94 x 106
Total Assay Volume (µL) 100 100 25
µL/Well User purified reagent 10 10 2.5
mL Purified rgt/plate 1 1 1
mL Total purified rgt 5 375 94
30,000 Compounds Traditional Assay 96-Well HTS
Plates/Day 1 40
Total Plates 5 375
Cells/Day 1 x 106 40 x 106
Total Cells 5 x 106 375 x 106
Total Assay Volume (µL) 100 100
µL/Well User purified reagent 10 10
mL Purified rgt/plate 1 1
mL Total purified rgt 5 375
Traditional Assay
Plates/Day 1
Total Plates 5
Cells/Day 1 x 106
Total Cells 5 x 106
Total Assay Volume (µL) 100
µL/Well User purified reagent 10
mL Purified rgt/plate 1
mL Total purified rgt 5
13
Consider Reagent Stability Compare traditional
assays with HTS assays
Traditional assay Reagent stability 30-minutes
to manually setup plate 2-hr incubation for an
end-point measurement 5-minutes to read
plate Reagents need to be stable for up to 2.5hr
HTS assay Reagent stability 5-minutes to setup
each plate 2-hr incubation for an end-point
measurement 5-minutes to read plate, 40
plates/run total Reagents need to be stable from
first to last plate Up to 3 hrs for plate setup,
3hr read time Up to 8 hrs from start to
finish Can remaining reagents be reused
14
Consider Availability of appropriate positive
and negative controls
Traditional assessments of assay quality
S/B Mean PosCtl / Mean NegCtl
S/N (Mean PosCtl - Mean NegCtl) / StdDev NegCtl
Assay 1 Ve mean 50, Ve- mean 10 S/B 5, S/N
13
Assay 2 Ve mean 112, Ve- mean 10 S/B 11,
S/N 39
15
Availability of appropriate positive and negative
controls
Common HTS assessment of assay quality
(3 StdDev PosCtl) ( 3 StdDev
NegCtl) z 1 - ---------------------------------
--------------------------
Mean PosCtl - Mean NegCtl
16
Availability of appropriate positive and negative
controls
Common HTS assessment of assay quality
Separation Band
Ve mean 50, Ve- mean 10 S/B 5, S/N 13,
z 0.5
17
Availability of appropriate positive and negative
controls
Common HTS assessment of assay quality
Ve mean 112, Ve- mean 10 S/B 11, S/N 39,
z 0.0
18
Availability of appropriate positive and negative
controls
Common HTS assessment of assay quality
z 1 An ideal assay
1 gt z 0.5 A good assay
0.5 gt z gt 0 Assay will require replicates
z 0 Yes/No type assay
z 0.5
z 0.1
19
Assay Issues to be Addressed Prior to Testing the
First Compound
Plate Uniformity and Signal Variability
Testing Critical testing of an assay system
prior to screening for all assays run in the
core These assays test the performance of the
following controls Maximum signal reference
(highest assay end point) Minimum signal
reference (background/lowest assay end
point) Midrange signal reference (signal
variability assessment)
20
Assay Issues to be Addressed Prior to Testing the
First Compound
For all assays run in the core, similar results
must be obtained over three separate days
(independent experiments in triplicate) using all
equipment and compound vehicle that will be
used during the screen. Acceptance
criteria Intraplate variability No apparent
edge effects or drift CVmax and CVmin lt
20 z 0.4 Interplate and Inter-Day
variability Midrange control lt 2-fold within a
single day Midrange control lt 2-fold across any
two days
21
What Are These Small Molecules We Test?
They are not DNA, RNA, or protein
macromolecules Practical Definition An organic
molecule of less than 1000 Daltons Typically in
the range of 300-700 Daltons Small organic
molecules made by living organisms (e.g.,
natural products) Small organic molecules made
by chemists (e.g., organic compounds, RNAi) In
all cases one is looking for a small drug-like
organic molecule that displays a biological
activity (e.g., agonist, antagonist) with the
target of interest.
22
How Does One Select a Library to Screen?
Random Selection Random high throughput
screening Little is known about the target Few
or no active compounds as guides
Computational Chemistry/Virtual
Screening Creation of Focused
Libraries Requires prior knowledge about
target Active compounds, 3D-Structure Sequence
homology
Prior Experience Library successfully used for
similar or related targets
23
Core Lab HTS Hit Guidelines
On each screening day, z-factor (controls) is
evaluated for every plate Controls must meet
original acceptance criteria previously defined
for the assay Only outliers dropped are those
created because of assay error or those gt 3 SD
from mean for all of that particular control No
more than 10-25 of a particular control will
be dropped for any particular plate
z 0.5
z 0.1
24
Core Lab HTS Hit Guidelines
Active compounds are those outside 3 SD from the
mean for all test agents on a valid plate. If
replicates are performed, a test agent must be
active on 66 of all replicates to be
classified as active. Hits are those active
compounds that demonstrate concentration
response upon reorder and retest.
25
Helpful References
Inglese J, Johnson RL, Simeonov A, Xia M, Zheng
W, Austin CP, Auld DS. High-throughput screening
assays for the identification of chemical
probes. Nat Chem Biol. 2007 Aug3(8)466-79.
Zhang JH, Chung TD, Oldenburg KR. A Simple
Statistical Parameter for Use in Evaluation and
Validation of High Throughput Screening Assays. J
Biomol Screen. 19994(2)67-73.
Iversen PW, Eastwood BJ, Sittampalam GS, Cox
KL. A comparison of assay performance measures
in screening assays signal window, Z' factor,
and assay variability ratio. J Biomol Screen.
2006 Apr11(3)247-52.
Inglese J, Shamu CE, Guy RK. Reporting data from
high-throughput screening of small-molecule
libraries. Nat Chem Biol. 2007 Aug3(8)438-41.
National Institutes of Health (NIH) Chemical
Genomics Center (NCGC), a member of the
Molecular Libraries Probe Production Center
Network. http//ncgc.nih.gov/
26
Cliff Stephan, Ph.D.
Postdoctoral training Cardiovascular Division
B.A. Chemistry and Molecular Biology Ph.D.
Pharmacology
Research Instructor, Cardiovascular Department,
Hypertension Division
Director of High Throughput Screening
Head of the John S Dunn Central Screening Core
Laboratory
27
(No Transcript)