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SESSION IIEarly Phases of Drug Discovery Chair — Kurt R. Brunden, PhD, University of Pennsylvania Session OverviewKurt 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 PotencyKurt R. Brunden, PhD, University of Pennsylvania
Importance of ToxicologyJohn E. Sagartz, DVM, PhD, DACVP, Seventh Wave Laboratories
Jim Inglese, Ph.D.National Center for Advancing Translational Sciences
National Human Genome Research InstituteNational Institutes of Health
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
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
• 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
High Throughput Screening
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
• 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%)• 1536-well: 32 x 48, 1,408 samples, 128 ctrl (8.3%)
row
column
Low volume microtiter plates for HTS
96-well plate
384-well plate
1536-well plate
For the same % of plate get 16x more control wells—allows full ctrl titrations (e.g., 128 ctrl wells vs. 8)
50 L
20 L
4 L
1/10 tear drop
test
6.8 mm 3.6
mm 1.4 mm
96
384
1536
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology
Parallel processing of assays
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
@ 200* microtiter plates per 24 hrs:
• 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
Reagent and Compound Delivery Systems
Buffers and cells
Library compounds
Compound combinations
1/500 tear drop
1/2500 tear drop
test
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology
Detectors and data analysis
Signal detection modalities & plate types
High content information
test
• Detection of Biological Responses, primarily created by a fluorescent or luminescent mechanism, is the principle HTS detection modality.
• ligand binding– competition binding
• enzymatic activity– biochemical or cellular
• ion or ligand transport– ion-sensitive or membrane potential dyes– 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.
Detectors and data analysis
Signal detection modalities & plate types
High content informationData analysis
“Candidate Hits”
test
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology
• Detection of Biological Responses, primarily created by a fluorescent or luminescent mechanism, is the principle HTS detection modality.
Categories of chemical libraries used in HTS
Nature 448: 645-6, 2007
Huang, R. et al. 2011 Sci Trans Med 3 (80) ; Kawamura, T. et al. 2011 BMC 19 4377
• 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
– 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)
Example chemical libraries used in drug discovery
Library Category SizeSigma Library of Pharmaceutically Active Compounds (LOPAC)
Pharmacologically active (drugs and probes)
1,208
ChemBridge Fragment Set high aqueous solubility (~3 mM)(Low MW (≤ 300), and cLogP( ≤ 3))
5,000
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 libraries) to high diversity
100s to >100K
NIH Molecular Libraries Small Molecule Repository (ML SMR)
Diverse collection: procured, QC’ed, stored and distributed to 10 network labs
>400K→500K
Pfizer compound file Large pharma collection. Outsourced from ArQule, ChemRx, ChemBridge and Tripos
>2x106
Malaria Tool Box Indication targeted 10KGlaxoSmithKline PKI Published Set Gene family targeted (kinases) 367Hoffman 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
• 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.
Special requirements of HTS assaysParameter ‘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 monthsMeasurements as endpoint, multiple time points, or continuous
Minutes to hoursMeasurements typically endpoint, but also pre-read and kinetic
Output formats Plate reader, radioactivity, size separation, object enumeration, images interpreted by human visual inspection
Plate reader: mostly fluorescence, luminescence and 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
For a review see: Inglese et al. 2007 Nat Chem Biol 3(8) 466
Stable clonal cells Transiently transfected cells
*
Top conc. only
High (50 M) low
1 : 2 dilution
50 M
Top conc. only
High (50 M) low
1 : 2 dilution
50 M
S-W Jang
Stable clonal cells Transiently transfected cells
96-well plate
1536-well plate
Special requirements of HTS assaysParameter ‘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 monthsMeasurements as endpoint, multiple time points, or continuous
Minutes to hoursMeasurements typically endpoint, but also pre-read and kinetic
Output formats Plate reader, radioactivity, size separation, object enumeration, images interpreted by human visual inspection
Plate reader: mostly fluorescence, luminescence and 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
*
*
Typical 1536-well plate assay protocol
Biochemical assay - Tau Filbrillization
Cell-based assay – CMT1A
Target-focused design
Pathway-focused design
• 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.
Automation engineering and informatics
+
=
Libraries of pure compoundsAssaysi. diverse scaffoldsii. elaborated scaffold (e.g., targeted)iii. bioactive (e.g., approved drugs)iv. natural products & derivatives
i. isolated molecular targetii. targeted cell pathwaysiii. reconstituted systemsiv. cell-based phenotypic v. model organism/parasite
For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology
Apparent Activity in High-Throughput Screening: Origins of Compound-Dependent Assay Interference
Phenomenon Hallmark Example Diagnostic
Inner filer effect
Enzyme or target-independentColored / pigmented compounds
Aggregation potential
Detergent-dependentSteep Hill slope
Redox activity Buffer component-dependent
Fluorescence Sample fluorescence overlaps λEM
Reporter pharmacology
Reporter-dependent SAR
Thorne, N. et al. 2010 Curr Opin Chem Biol 14:315-324
The HTS Parallax View
Translational research in collaboration with Charcot-Marie-Tooth Association http://www.cmtausa.org
pmp22
pmp22
pmp22 pmp22Duplication (CMT1A)Deletion (HNPP)
Curr Opin Neurol. 2004 Oct;17(5):579-85
Normal conduction velocity 55-60 m/s
Charcot-Marie-Tooth (CMT) Disease
15-25 m/s
Nature Rev Neurosci 4, 714-726 & 6, 683-690 Trends in Genetics, 1998,Oct; 14(10): 417-422
Goal: Develop chemical agents that transcriptionally repress the expression of the PMP22 gene
RNase protection assay
J Neurosci Res. 2002 Aug 15;69(4):497-508 Jones et al, J Neurosci, 2011
Stably transfected into S16 (Rat Schwann cells) cell lineConstruct
Locus
Cell line PMP22 levels Reporter response to Sox10 KD
21
Jang, S.-W. et al, submitted
CONTEXT SIGNATURE-BASED OUTPUT
Bioluminescence & fluorescence HTS compatible pathway and network assay formats
β-lactamase reporter geneFirefly luciferase reporter gene
RG
“Target” τa
b
cd
e f
Combining cross-validating orthogonal assays with qHTS in a drug repurposing experiment
• 1536-well plates, inter-plate dilution series• Assay volumes 2-5 μL
• Assay concentration ranges over 4 logs (high:~ 60 μM) • Curve fitting classification (Class 1-4)
• Establish nascent SAR, pharmacological dependence
• Reconstruct concentration-response data
B
C
Inglese et al. (2006) PNAS 103, 11473-11478
D • Combined with cross-validating orthogonal assays should allow rapid identification of biologically relevant modulators
FLuc Lac
• 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)
E
A
Selection and profile of active drugs identified from qHTSH
TS
Low
thr
ough
put
A 1536-well plate HTS assay for Tau Assembly
• 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
Biochemical assay for protein-protein interaction - Target-focused design
---
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-
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30 uM Thioflavin T
40 uM Heparin
15 uM Tau P31L
Thioflavin T
0.12 uM Tau Alexa 594---
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-
---------
-
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--
-- -
--
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30 uM Thioflavin T
40 uM Heparin
15 uM Tau P31L
Thioflavin TThioflavin T
0.12 uM Tau Alexa 594
Crowe, A. (2009) Biochemistry 48, 7732-7745
*Class -1 and -2.1 actives
Tau assay qHTS performance metrics
3s+ 3s -
S:B
Class -2
ThT
FP
Class -1 Class -3
qHTS Titration-response plots of tau inhibitors from ~292,000 compounds of the NIH Molecular Libraries SMR
Crowe, A. (2009) Biochemistry 48, 7732-7745
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
HTS: In the midst of translation
bioactivity measurement
X*
*X = 1 or other
cpd optimization
2
3
n
model systems cpd profile
assays
active cpdslibrary
HTS
NIH Molecular Libraries ProgramProbe Production Centers Network (MLPCN)
mli.nih.gov
NIH Programs to Aid Drug Discovery
Clinical Trials
Lead Lead
Optim. Preclinical
Devel. Hit HTS
TargetFDA
approval
(probe)
Assay Devel.
Assay Development for Rare and Neglected Diseases(will appear on NCATS website)
NIH Therapeutics for Rare and Neglected Disease(TRND)
trnd.nih.gov
National Institutes of Health (NCGC)Scripps Research InstituteThe Sanford-Burnham Institute The Broad InstituteJohns Hopkins UniversitySouthern Research InstituteUniversity of New Mexico University of KansasVanderbilt University
NCATS Contact Info
• To inquire about assay development, screening or submitting chemical libraries to NCATS contact: [email protected]
• More info available at• http://www.ncats.nih.gov/• http:/mli.nih.gov• http:/trnd.nih.gov
Compound Optimization After HTS – Beyond Potency
Kurt R. Brunden, Ph.D.University of Pennsylvania
6th Drug Discovery for Neurodegeneration ConferenceNew York, NY
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