1. SESSION II
Early Phases of Drug Discovery
Chair — Kurt R. Brunden, PhD, University of Pennsylvania
Session Overview
Kurt R. Brunden, PhD, University of Pennsylvania
Basics of High Throughput Screening (HTS)
James Inglese, PhD, National Institutes of Health Chemical Genomics Center
Compound Optimization after HTS: Beyond Potency
Kurt R. Brunden, PhD, University of Pennsylvania
Importance of Toxicology
John E. Sagartz, DVM, PhD, DACVP, Seventh Wave Laboratories

2. Basics of High Throughput Screening: Bridging
Chemistry and Biology
6th DRUG DISCOVERY FOR NEURODEGENERATION:
An Intensive Course on Translating Research into Drugs
February 12-14, 2012, New York, NY
Jim Inglese, Ph.D.
National Center for Advancing Translational Sciences
National Human Genome Research Institute
National Institutes of Health

3. Outline
• Overview of HTS process
– Currently practiced across majority of industry & academia
– Spectrum of chemical libraries in use
– Design of assays compatible with HTS
– Issues arising at the intersection of chemical libraries with HTS
assays
• Case studies
– PNS disease
• phenotypic assay and approved drug screening
– CNS disease
• target-based screening of a large chemical library
• Access to NIH Drug Discovery & Development Resources

4. High Throughput Screening
• High Throughput Screen (HTS): An iterative testing of
different substances in a common assay generally for
>10,000 samples per day.
• Assays designed for HTS attempt to integrate
biological fidelity with high sensitivity assay &
screening technologies test
• The configuration and nature of the assay formats are critical to the HTS
experiment and must be coordinated with the nature of the compounds
tested and subsequent assays that evaluate biological relevance/mechanism
of action.
For a review see: Inglese et al. 2007 Nature Chem Biol 3, 466-479

5. Low volume microtiter plates for HTS
• To increase efficiency assay volumes are reduced:
• 96 well: 8 x 12, 88 samples, 8 ctrl (8.3%)
• 384-well: 16 x 24, 352 samples, 32 ctrl (8.3%) column
• 1536-well: 32 x 48, 1,408 samples, 128 ctrl (8.3%)
row
96-well plate test
50 L
96
384-well plate 20 L
384
1536-well plate
1536
6.8 mm
4 L 3.6 mm
1.4 mm
1/10 tear drop
For the same % of plate get 16x more control wells—allows full ctrl titrations
(e.g., 128 ctrl wells vs. 8) For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology

6. Parallel processing of assays
@ 200* microtiter plates per 24 hrs:
Plate format Sample wells/day Time to screen
1 MM samples
96-well 19,200 3.2 months
384-well 76,800 3½ weeks
1,536-well 307,200 3 ½ days

7. Reagent and Compound Delivery Systems
• Typical assay volumes in a 1536-well plate (2-8 L) require:
• reagent addition volumes of 0.5 – 5 L
• compound addition volume of ~20 nL
Buffers and cells
1/500 tear drop
test
Library compounds
1/2500
Compound combinations tear drop
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology

8. Detectors and data analysis
• Detection of Biological Responses, primarily created
by a fluorescent or luminescent mechanism, is the
principle HTS detection modality.
Signal detection modalities & plate types
test
• ligand binding
– competition binding
• enzymatic activity
– biochemical or cellular
• ion or ligand transport
– ion-sensitive or membrane potential dyes
High content information – current measurements
• protein-protein interactions
– biochemical or cellular
• gene transcription
‒ mRNA
• cellular signaling & metabolism
– reporter gene
– second messenger
– MS-based metabolite measurements
• phenotypic
– cell viability
– protein redistribution
– multiparametric imaging
– etc.

9. Detectors and data analysis
• Detection of Biological Responses, primarily created
by a fluorescent or luminescent mechanism, is the
principle HTS detection modality.
Signal detection modalities & plate types
test
High content information
Data analysis
“Candidate Hits”
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology

10. Categories of chemical libraries used in HTS
• Library membership size
– small (~100’s-1000’s), moderate (>10K), large (100K to several million)
• Biologically active (limited in number)
• Synthetic bioactives & natural products
• Approved drugs
– Complex mixtures
• Natural product extracts Nature 448: 645-6, 2007
– culturable / non-culturable
• Pooled synthetic libraries
• Synthetic extracts
– Privileged scaffold-based libraries
• Untested analogs of synthetic drugs or natural products
– Benzodiazapines, imidazoly pyrimidines
– ‘Unnatural natural product’ library
• Biologically uncharacterized (vast in number)
– Low diversity, high density
• combinatorial chemistry-derived libraries
– Consolidated samples/collections-- extensive structural diversity
• Pharma Libraries
• Molecular Libraries Small Molecule Repository (see PubChem)
Huang, R. et al. 2011 Sci Trans Med 3 (80) ; Kawamura, T. et al. 2011 BMC 19 4377

11. Example chemical libraries used in drug discovery
Library Category Size
Sigma Library of Pharmaceutically Active Pharmacologically active 1,208
Compounds (LOPAC) (drugs and probes)
ChemBridge Fragment Set high aqueous solubility (~3 mM) 5,000
(Low MW (≤ 300), and cLogP( ≤ 3))
TimTech Natural compound library (NPL400) Purified natural products 480
Remodeled natural products Diversity oriented synthesis (DOS) ~2,000
National Toxicology Program (NTP1,408) Toxic agents 1,408 -10K
Commercial screening libraries Range from low scaffold diversity (e.g., CC 100s to >100K
libraries) to high diversity
NIH Molecular Libraries Small Molecule Diverse collection: procured, QC’ed, stored >400K
Repository (ML SMR) and distributed to 10 network labs →500K
Pfizer compound file Large pharma collection. Outsourced from >2x106
ArQule, ChemRx, ChemBridge and Tripos
Malaria Tool Box Indication targeted 10K
GlaxoSmithKline PKI Published Set Gene family targeted (kinases) 367
Hoffman LaRoche PKI Set Gene family targeted (kinases) 235
NIH Pharmaceutical Collection Approved drugs >3000
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology

12. • HTS Assay: An efficiently-designed experiment
measuring the effect of a substance on a
biological process of interest.
Spend the time developing the
right assay(s); the cost of failure
appears to increase
exponentially the further it occurs
from the start of a program.

13. Special requirements of HTS assays
Parameter ‘Bench top’ HTS
Protocol May be complex with numerous steps, aspirations, washes Few (5-10) steps, simple operations, addition only preferred
Assay volume 0.1 mL to 1 mL <1 L* to 100 L
Reagents Quantity often limited, batch variation acceptable, may be unstable Sufficient quantity, single batch, must be stable over
* prolonged period
Reagent handling Manual Robotic
Variables Many: e.g., time, substrate/ligand concentration, compound, cell type Compound**, compound concentration
Assay container Varied: tube, slide, microtiter plate, Petri dish, cuvette, animal, etc. Microtiter plate
Time of measurement Milliseconds to months Minutes to hours
Measurements as endpoint, multiple time points, or continuous Measurements typically endpoint, but also pre-read and
kinetic
Output formats Plate reader, radioactivity, size separation, object enumeration, Plate reader: mostly fluorescence, luminescence and
images interpreted by human visual inspection absorbance
Reporting format “Representative” data; statistical analysis of manually curated dataset Automated analysis of all data using statistical criteria
Notes: *special reagent dispensers required; **ideally available in mg quantity with analytical verification of structure and purity
Stable clonal cells Transiently transfected cells
For a review see: Inglese et al. 2007 Nat Chem Biol 3(8) 466

14. Stable clonal cells Transiently transfected cells
96-well plate
1536-well plate
High (50 M) low 50 M High (50 M) low 50 M
1 : 2 dilution Top conc. only 1 : 2 dilution Top conc. only
S-W Jang

15. Special requirements of HTS assays
Parameter ‘Bench top’ HTS
* Protocol
Assay volume
May be complex with numerous steps, aspirations, washes
0.1 mL to 1 mL
Few (5-10) steps, simple operations, addition only preferred
<1 L* to 100 L
Reagents Quantity often limited, batch variation acceptable, may be unstable Sufficient quantity, single batch, must be stable over
prolonged period
Reagent handling Manual Robotic
Variables Many: e.g., time, substrate/ligand concentration, compound, cell type Compound**, compound concentration
Assay container Varied: tube, slide, microtiter plate, Petri dish, cuvette, animal, etc. Microtiter plate
Time of measurement Milliseconds to months Minutes to hours
Measurements as endpoint, multiple time points, or continuous Measurements typically endpoint, but also pre-read and
kinetic
Output formats Plate reader, radioactivity, size separation, object enumeration, Plate reader: mostly fluorescence, luminescence and
images interpreted by human visual inspection absorbance
Reporting format “Representative” data; statistical analysis of manually curated dataset Automated analysis of all data using statistical criteria
Notes: *special reagent dispensers required; **ideally available in mg quantity with analytical verification of structure and purity
* •
•
Eppendorf tubes
vortex
• centrifuge tubes
• light sensitive materials
– light box
– dark room
• separation
– Hamilton syringe
• re-suspension
• SDS-PAGE separation
– dry gel
• expose to x-ray film
• densitometer
Inglese, Koch, Caron, & Lefkowitz, 1992 Nature 359 For a review see: Inglese et al. 2007 Nat Chem Biol 3(8) 466

16. Typical 1536-well plate assay protocol
Biochemical assay - Tau Filbrillization
Target-focused design
Cell-based assay – CMT1A
Pathway-focused design

17. • Technologically-enabled HTS creates an efficient interface between biological
assays and chemical libraries that allow the rapid identification and profiling of
wide-ranging chemotypes that modulate individual gene products or
cellular/organism phenotypes on a large scale.
Assays Libraries of pure compounds
i. isolated molecular target
ii. targeted cell pathways i. diverse scaffolds
iii. reconstituted systems ii. elaborated scaffold (e.g., targeted)
iv. cell-based phenotypic iii. bioactive (e.g., approved drugs)
v. model organism/parasite iv. natural products & derivatives
+
=
Automation engineering and informatics
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology

18. Apparent Activity in High-Throughput Screening: Origins of
Compound-Dependent Assay Interference
Phenomenon Hallmark Example Diagnostic
Enzyme or target-independent
Inner filer
effect Colored / pigmented
compounds
Aggregation Detergent-dependent
potential Steep Hill slope
Redox activity Buffer component-dependent
Sample fluorescence overlaps
Fluorescence
λEM
Reporter
Reporter-dependent SAR
pharmacology
Thorne, N. et al. 2010 Curr Opin Chem Biol 14:315-324

19. The HTS Parallax View

20. Translational research in collaboration with Charcot-Marie-
Tooth Association http://www.cmtausa.org
pmp22
pmp22
pmp22 pmp22
Deletion (HNPP) Duplication (CMT1A)
Normal conduction velocity
55-60 m/s
Charcot-Marie-Tooth (CMT)
Disease
15-25 m/s
Curr Opin Neurol. 2004 Oct;17(5):579-85 Trends in Genetics, 1998,Oct; 14(10): 417-422 Nature Rev Neurosci 4, 714-726 & 6, 683-690

21. Goal: Develop chemical agents that transcriptionally repress
the expression of the PMP22 gene
Locus
Stably transfected into S16 (Rat
Construct Schwann cells) cell line
CONTEXT SIGNATURE-BASED OUTPUT
Cell line PMP22 levels Reporter response to Sox10 KD
 

RNase protection assay 21
J Neurosci Res. 2002 Aug 15;69(4):497-508 Jones et al, J Neurosci, 2011 Jang, S.-W. et al, submitted

22. Bioluminescence & fluorescence HTS compatible
pathway and network assay formats
“Target” e f
a τ
c RG
d
b
Firefly luciferase reporter gene β-lactamase reporter gene

23. Combining cross-validating orthogonal assays with qHTS
in a drug repurposing experiment
• Assay concentration ranges over 4 logs (high:~ 60 μM)
• Curve fitting classification (Class 1-4)
A • 1536-well plates, inter-plate dilution series C • Establish nascent SAR, pharmacological
• Assay volumes 2-5 μL dependence
• Combined with cross-validating orthogonal
D assays should allow rapid identification of
biologically relevant modulators
 FLuc Lac

B • Reconstruct
concentration-
response data
E
Counter screen for overt cellular toxicity
• (NOT a
ADME/Tox consideration, rather to control for a technical
artifact for loss –of –signal cell-based assays)
Inglese et al. (2006) PNAS 103, 11473-11478

24. Selection and profile of active drugs identified from qHTS
HTS
Low throughput

25. A 1536-well plate HTS assay for Tau Assembly
Biochemical assay for protein-protein interaction - Target-focused design
Thioflavin T
30 uM Thioflavin T
15 uM Tau P31L - - - -
-
-
- -
-
-
- - -
-
--
- --
0.12 uM Tau Alexa 594
-
- -
- -
-
-
-
-
40 uM Heparin
--
-
-
-
-
• Target: Tau (oligomerization and/or fibrillization)
• Assay: fibrillization of a truncated tau fragment monitored by
complementary thioflavin T fluorescence and FP of
substiochiometricly labeled tau
Crowe, A. (2009) Biochemistry 48, 7732-7745

26. Tau assay qHTS performance metrics
*Class -1 and -2.1 actives

27. qHTS Titration-response plots of tau inhibitors from
~292,000 compounds of the NIH Molecular Libraries SMR
Class -1 Class -2 Class -3
ThT
FP
Crowe, A. (2009) Biochemistry 48, 7732-7745

28. Derivation of SAR and candidate selection
• All FP class 1 and 2.1 compounds were grouped into clusters comprised of
shared core structural elements
• 42 series with no liabilities
• Inconsistent activity (inactive in ThT assay)
• Fluorescence/absorbance (changes in total fluorescence in FP assay, spectroscopic profiling)
• Promiscuous aggregators (cruzain activity ± detergent)
• Low potency/efficacy
• Low percentage of active compounds
• Previously described classes of inhibitors:
• Aminothienopyridazines (ATPZs): a novel scaffold with promising drug-like
features and biochemical properties.
• E.g., no significant effect on tau-mediated tubulin polymerization
Crowe, A. (2009) Biochemistry 48, 7732-7745

29. HTS: In the midst of translation
HTS
library bioactivity measurement active cpds
assays
2
3 X*
n
*X = 1 or other
model systems cpd optimization cpd profile

30. NIH Programs to Aid Drug Discovery
Assay Lead Preclinical Clinical
HTS Hit Lead
Devel. (probe) Optim. Devel. Trials
FDA
Target
approval
Assay Development for Rare and Neglected Diseases
(will appear on NCATS website)
NIH Molecular Libraries Program
Probe Production Centers Network (MLPCN)
National Institutes of Health (NCGC)
mli.nih.gov
Scripps Research Institute
The Sanford-Burnham Institute
The Broad Institute
Johns Hopkins University NIH Therapeutics for Rare and Neglected Disease
Southern Research Institute (TRND)
University of New Mexico
University of Kansas trnd.nih.gov
Vanderbilt University

31. NCATS Contact Info
• To inquire about assay development, screening or
submitting chemical libraries to NCATS contact:
jinglese@mail.nih.gov
• More info available at
• http://www.ncats.nih.gov/
• http:/mli.nih.gov
• http:/trnd.nih.gov

32. Compound Optimization After HTS
– Beyond Potency
Kurt R. Brunden, Ph.D.
University of Pennsylvania
6th Drug Discovery for Neurodegeneration Conference
New York, NY

33. Overview
• Discuss systems and assays that can be reasonably
implemented by academic groups for CNS drug
discovery.
• Assumes existing target-specific potency and selectivity
assays (e.g., related receptors or enzymes).
• Discussion topics:
– ADME (Absorption, Distribution, Metabolism and Excretion)
• Solubility
• Pharmacokinetics
• Metabolism
– Toxicology
• In Vitro assays
• Rodent tolerability studies