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 HTS and Lead Id Lead Opt Basic Research 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 > 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 >1,000,000 data points/day HTS in the Dunn Screening Core the potential of screening 100’s to >10,000 data points/day following Pharma industry standards

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 Traditional style assays Multiple steps, more manipulations, slower throughput Examples: Traditional binding assays Traditional sandwich ELISA

8. Compare Traditional Assays with HTS Assays

9. Key factors for successful HTS • time/well • wells/day • screens/year • project time Time HTS Screen Quality Costs • few false positives • few false negatives • S:N,SW,z’-Factor • Validated ‘Hits’ • reagents • consumables • instrumentation • personnel

10. HTS: An Iterative Process Research groups Target Id and Validation Develop Primary and Secondary assays Define criteria for active compounds Direct ‘Hit’ improvement process • HTS Group • Perform Primary Screen • Purpose: Identify a starting place • Method: Interrogate libraries of • compounds/genes 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

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’ = 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 < 20% z’ ≥ 0.4 Interplate and Inter-Day variability: Midrange control < 2-fold within a single day Midrange control < 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 > 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 Aug;3(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. 1999;4(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 Apr;11(3):247-52. Inglese J, Shamu CE, Guy RK. Reporting data from high-throughput screening of small-molecule libraries. Nat Chem Biol. 2007 Aug;3(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