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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

Session 2 part 1

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Page 1: Session 2 part 1

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    

Page 2: Session 2 part 1

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

Page 3: Session 2 part 1

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

Page 4: Session 2 part 1

• 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

Page 5: Session 2 part 1

• 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

Page 6: Session 2 part 1

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:

Page 7: Session 2 part 1

• 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

Page 8: Session 2 part 1

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.

Page 9: Session 2 part 1

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.

Page 10: Session 2 part 1

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)

Page 11: Session 2 part 1

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

Page 12: Session 2 part 1

• 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.

Page 13: Session 2 part 1

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

*

Page 14: Session 2 part 1

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

Page 15: Session 2 part 1

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

*

*

Page 16: Session 2 part 1

Typical 1536-well plate assay protocol

Biochemical assay - Tau Filbrillization

Cell-based assay – CMT1A

Target-focused design

Pathway-focused design

Page 17: Session 2 part 1

• 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

Page 18: Session 2 part 1

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

Page 19: Session 2 part 1

The HTS Parallax View

Page 20: Session 2 part 1

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

Page 21: Session 2 part 1

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

Page 22: Session 2 part 1

Bioluminescence & fluorescence HTS compatible pathway and network assay formats

β-lactamase reporter geneFirefly luciferase reporter gene

RG

“Target” τa

b

cd

e f

Page 23: Session 2 part 1

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

Page 24: Session 2 part 1

Selection and profile of active drugs identified from qHTSH

TS

Low

thr

ough

put

Page 25: Session 2 part 1

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

---

--- -

-

-----

-

---

---

---

30 uM Thioflavin T

40 uM Heparin

15 uM Tau P31L

Thioflavin T

0.12 uM Tau Alexa 594---

--- -

-

---------

-

---

---

--- -

--

-- -

--

---

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

Page 26: Session 2 part 1

*Class -1 and -2.1 actives

Tau assay qHTS performance metrics

3s+ 3s -

S:B

Page 27: Session 2 part 1

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

Page 28: Session 2 part 1

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

Page 30: Session 2 part 1

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

Page 31: Session 2 part 1

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

Page 32: Session 2 part 1

Compound Optimization After HTS – Beyond Potency

Kurt R. Brunden, Ph.D.University of Pennsylvania

6th Drug Discovery for Neurodegeneration ConferenceNew York, NY

Page 33: Session 2 part 1

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