Session 2 part 1

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  • -This presentation is meant to be a short tutorial on assays that can be reasonably implemented in academic drug discovery labs that will facilitate compound optimization in important dimensions besides potency and selectivity. -In particular, we will discuss testing of compounds to verify that they are suitable for in vivo efficacy testing in rodent disease models. In addition, there are certain basic toxicological assessments that can be performed that will help determine whether a compound has the potential to be an IND candidate.-It is assumed that the academic laboratory will have in place adequate potency assays for the target of choice, as well as assays for highly related targets (e.g., if studying a serotonin receptor, functional or binding assays to related serotonin receptors). -It is also assumed that the laboratory has a source of compounds, either through collaboration or an internal chemistry effort.-The aspects of compound characterization and optimization that I will cover in this presentation are ADME, in vitro safety toxicology assays, and rodent tolerability studies.
  • Session 2 part 1

    1. 1. SESSION IIEarly Phases of Drug DiscoveryChair — Kurt R. Brunden, PhD, University of PennsylvaniaSession OverviewKurt R. Brunden, PhD, University of PennsylvaniaBasics of High Throughput Screening (HTS)James Inglese, PhD, National Institutes of Health Chemical Genomics CenterCompound Optimization after HTS: Beyond PotencyKurt R. Brunden, PhD, University of PennsylvaniaImportance of ToxicologyJohn E. Sagartz, DVM, PhD, DACVP, Seventh Wave Laboratories
    2. 2. Basics of High Throughput Screening: BridgingChemistry 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. 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. 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. 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 dropFor 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. 6. Parallel processing of assays@ 200* microtiter plates per 24 hrs: Plate format Sample wells/day Time to screen 1 MM samples96-well 19,200 3.2 months384-well 76,800 3½ weeks1,536-well 307,200 3 ½ days
    7. 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. 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. 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. 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. 11. Example chemical libraries used in drug discovery Library Category SizeSigma Library of Pharmaceutically Active Pharmacologically active 1,208Compounds (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 480Remodeled natural products Diversity oriented synthesis (DOS) ~2,000National Toxicology Program (NTP1,408) Toxic agents 1,408 -10KCommercial screening libraries Range from low scaffold diversity (e.g., CC 100s to >100K libraries) to high diversityNIH Molecular Libraries Small Molecule Diverse collection: procured, QC’ed, stored >400KRepository (ML SMR) and distributed to 10 network labs →500KPfizer compound file Large pharma collection. Outsourced from >2x106 ArQule, ChemRx, ChemBridge and TriposMalaria Tool Box Indication targeted 10KGlaxoSmithKline PKI Published Set Gene family targeted (kinases) 367Hoffman LaRoche PKI Set Gene family targeted (kinases) 235NIH Pharmaceutical Collection Approved drugs >3000 For a review see: Inglese & Auld 2008 Wiley Encyclopedia of Chemical Biology
    12. 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. 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. 14. Stable clonal cells Transiently transfected cells 96-well plate 1536-well plateHigh (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. 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. 16. Typical 1536-well plate assay protocolBiochemical assay - Tau Filbrillization Target-focused designCell-based assay – CMT1A Pathway-focused design
    17. 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. 18. Apparent Activity in High-Throughput Screening: Origins of Compound-Dependent Assay InterferencePhenomenon Hallmark Example Diagnostic Enzyme or target-independent Inner filer effect Colored / pigmented compoundsAggregation Detergent-dependent potential Steep Hill slopeRedox activity Buffer component-dependent Sample fluorescence overlapsFluorescence λEM Reporter Reporter-dependent SARpharmacology Thorne, N. et al. 2010 Curr Opin Chem Biol 14:315-324
    19. 19. The HTS Parallax View
    20. 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/sCurr 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. 21. Goal: Develop chemical agents that transcriptionally repressthe 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 21J Neurosci Res. 2002 Aug 15;69(4):497-508 Jones et al, J Neurosci, 2011 Jang, S.-W. et al, submitted
    22. 22. Bioluminescence & fluorescence HTS compatiblepathway and network assay formats “Target” e f a τ c RG d b Firefly luciferase reporter gene β-lactamase reporter gene
    23. 23. Combining cross-validating orthogonal assays with qHTSin 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. 24. Selection and profile of active drugs identified from qHTS HTS Low throughput
    25. 25. A 1536-well plate HTS assay for Tau AssemblyBiochemical 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 tauCrowe, A. (2009) Biochemistry 48, 7732-7745
    26. 26. Tau assay qHTS performance metrics*Class -1 and -2.1 actives
    27. 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 FPCrowe, A. (2009) Biochemistry 48, 7732-7745
    28. 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 polymerizationCrowe, A. (2009) Biochemistry 48, 7732-7745
    29. 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. 30. NIH Programs to Aid Drug Discovery Assay Lead Preclinical Clinical HTS Hit Lead Devel. (probe) Optim. Devel. Trials FDATarget 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. 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. 32. Compound Optimization After HTS – Beyond Potency Kurt R. Brunden, Ph.D. University of Pennsylvania6th Drug Discovery for Neurodegeneration Conference New York, NY
    33. 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

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