High-Throughput Screening

1. High-Throughput Screening

2. What is HTS ? Identification of one or more positive candidates extracted from a pool of 103 to 108 possible candidates based on specific criteria

3. High Throughput Screening • Library design (Library – Nature) • Assay technology (screening assay) • Laboratory Automation (high throughput) • The HTS work flow (target selection -> hit) • Data analysis of screening results

4. What do you use HTS for? To screen for all kind of novel biological active compounds (libraries): • Natural products • Combinatorial Libraries (peptides, chemicals…) • Biological libraries To screen MicroArrays such as: • DNA chips • RNA chips • Protein chips

5. How are Microarrays generated? DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput. Note: In the literature there exist at least two confusing nomenclature systems for referring to hybridization partners. Both use common terms: "probes" and "targets". According to the nomenclature recommended by B. Phimister of Nature Genetics, (volume 21 supplement pp 1 - 60, 1999) a "probe" is the tethered nucleic acid with known sequence, whereas a "target" is the free nucleic acid sample whose identity/abundance is being detected.

6. Microarray Technologies: • Format I: probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called DNA microarray, is widely considered as developed at Stanford University. A recent article by R. Ekins and F.W. Chu (Microarrays: their origins and applications. Trends in Biotechnology, 1999, 17, 217-218) seems to provide some generally forgotten facts. • Format II: an array of oligonucleotide (20~80-mer oligos) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. This method, "historically" called DNA chips, was developed at Affymetrix, Inc. , which sells its photolithographically fabricated products under the GeneChip® trademark. Many companies are manufacturing oligonucleotide based chips using alternative in-situ synthesis or depositioning technologies.

7. Steps in the design and implementation of the microarray experiment

8. Applications: • DNA Microarrays: Expression profiles, disease research (cancer), DNA sequencing, mutation analysis, gene discovery, diagnosis, drug discovery,… • RNA Microarrays: RNA-protein interactions, biological function of proteins, drug discovery,… • Protein Microarray (chips): Enzyme profiling, Protein-protein Interaction, Protein-ligand interaction ,...

9. Screening for novel compounds Past Natural product extracts Screening for novel biological active compounds Combinatorial libraries (organic chemistry) biological libraries (proteins, Virus,…) Future ?

10. Screening for novel compounds Screening of natural product extracts: -> Yes or No ??? In the past -> major source of diversity Originated mainly from plants and microorgansims Today -> 0.5% of microbes in soil tested -> same percentage of plants and microbes in ocean tested -> still a big potential of diversity !!!!! Difficulties: -> time and resource-intensive -> frequently not accessible by chemical synthesis (no derivates possible) -> chronic lack of success

11. Screening for novel compounds Screening of natural product extracts: -> Yes or No ??? Problems with screening different purity of extracts: -> crude extract: - many substances (complexity of function) - follow up isolation of active compound time consuming - many screens necessary to evaluate for tolerance in the screen (many different substances at different concentrations in the extract) -> partially purified: - still some substances (decreased complexity factor) - follow up isolation shortened - active substance lost partially (problem of concentration) or completely during purification process - substance not active any more – cofactor missing -> highly purified: - purification of individual substances from extract time consuming - high amounts of “start up” material necessary (problem with “unculturable” microbes) - wrong substances purified -> none of the candidates give a signal - active substance originally at too level to be purified (-> lost during purification process) - substance not active any more – cofactor missing -> Culture conditions determine what substances are produced !!!!

12. Screening for novel compounds • Screening of Libraries: • What is a library? • A pool of many related/similar substances (chemical compounds, proteins,…..) -> not natural occurring -> man made !!! • What libraries can we make – what are they used for? • -> Combinatorial libraries (chemical synthesis) • all kind of chemical compounds (including peptides) that can • be synthesized, used for ligand discovery, drug discovery, • interaction analysis, …. • -> Biological libraries (DNA libraries, RNA libraries, gene- • encoded libraries, protein libraries) • Microarrays, chip technology, screening for and engineering of • enzymes, antibodies, pathways, viruses, ligand discovery, • interaction analysis, …

13. Screening for novel compounds • Screening of Microorganisms -> for novel enzymes • -> by using gene probes -> cultivating • -> metagenomic screening -> non-cultivating • Gene probe technology -> hybridization technique • -> detection of presents and location of specific genes in organism • -> evaluate distribution of a gene among different species • -> taxonomic markers • -> search for novel enzymes in microbial isolates • Limitation: microbial strains can just be screened -> if cultivation possible !!!

14. Screening for novel compounds Screening of Microorganisms -> for novel enzymes 2. Metagenome -> screening of uncultivated microorganisms -> Fluorescence in situ hybridisation (FISH) -> 16S rRNA clone library -> metagenome ->16S rRNA clone library -> used to identify uncultivated microorganisms -> map their phylogenetic relationships (taxonomy)

15. Screening for novel compounds Screening of Microorganisms -> for novel enzymes -> Fluorescence in situ hybridization (FISH) -> can be used to detect and localize the presence or absence of specific DNAsequences on chromosomes. FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships. Bacterial FISH probes are often primers for the 16s rRNA region.

16. Screening for novel compounds Screening of Microorganisms -> for novel enzymes -> Metagenome -> extraction of DNA from all microorganisms present in isolate -> generate library or use as template for PCR Library -> BAC vector based (20-500 kbp insert) Can be screened by Microarray system: Target (immobilized) -> BAC clones Probe (labled) -> oligos directed to specific gene

17. Screening for novel compounds Screening of Microorganisms -> for novel enzymes Comparison of culturing and metagenome strategy Metagenome -> easier to clone complete pathways -> more difficult to make library

18. Screening for novel compounds Screening of Libraries: Library design: ! Size and Diversity -> direct impact on successful screening ! -> Targeted libraries -> derive from information gained on biomedical programs (find new lead structures) -> Focused libraries -> preselected subset of compounds based on virtual screening of a targeted library -> Discovery libraries -> randomly assembled (find new lead structures) 3 aspects of library design: -> combinatorial synthesis -> statistical considerations -> molecular recognition models

19. Screening for novel compounds Screening of Libraries: Library design: ! Size and Diversity -> direct impact on successful screening ! Limiting factors for library expansion: -> costs -> technical feasibility (storage, logistics) !!!Screening success -> frequently due to unexpected findings !!! (predictive capabilities still limited)

20. Screening for novel compounds What is a “lead structure” ? -> displays desired biological activity -> but does not yet combine all properties needed for therapeutic use Iterative cycles -> synthesis of structural analogs -> pharmacological testing -> suitable candidate for therapeutic use Properties a lead structure must have to become a pharmaceutical product: • Functional in vitro and in vivo (animal tests) • Reduced side-effects (understanding the structure-activity-relationship of the interaction between lead structure and receptor) • Chemical structure related to biological active molecule (optimal binding to receptor) • Accessible to chemical synthesis -> derivatisation • Substance and metabolic products not toxic • Novelty

21. Screening for novel compounds What is a “lead structure” ? Statins -> inhibits HMG-CoA reductase -> cholesterol Biosythesis Many companies have generated optimized products

22. Cholesterol Biosynthesis Blocker Potent blocker of HMG CoA reductase -> reduce level of synthesized cholesterol

23. Screening for novel compounds Assay Technology in HTS

24. Screening for novel compounds A -> Cell growth tests (cell-based assays) -> Phenotypic assays -> have problems to discriminate between pharmaceutical meaningful and toxic -> cellular response time can be long (min, hours, days) -> source of unspecific hits (compounds interfering with energy metabolism -> complexity!!!) B -> Tissue response -> targeted functional cell-based assay -> sensitivity and specificity towards receptor crucial for meaningful assay (Worse in A) -> recombinant cell lines (nowadays) make it possible to monitor functional activity -> response time (sec, hours) -> worse in A -> distinguishes between agonist and inhibitor C -> Enzyme test -> biochemical test -> best sensitivity -> can be run at high compound/solvent concentration -> discriminates between inhibitors and stimulators -> require recombinant gene expression and reconstitution of biological activity of protein (time consuming) -> great experimental freedom for varying conditions -> best for automated screening -> “fragment screening” (structural fragments used to identify low-affinity binding -> design larger compounds -> enhance affinity) Assay Technology in HTS

25. Screening for novel compounds A -> Cell growth tests (cell-based assays) -> Phenotypic assays Why? -> mechanism of action + relevance to disease -> complex targets -> necessary substrate and co-factor available at physiological conditions -> target difficult or expensive to express and purify -> sometime the fastest and least expensive approach Formats used: -> Reporter assay -> detect transcriptional regulation (luciferase, GFP, beta-galactosidase, …) -> Proximity assay -> screening for transporter targets (carrier) -> proliferation assay -> screens for targets that stimulate/inhibit cell growth -> assays to measure changes in gene expression (past: antibodies) -> today with microarray systems Implementation challenges: -> growth and adherence properties of cells -> solvent tolerance -> cytotoxicity -> stability of cellular phenotype Possible Future -> cell-based screenings on chips !!! Assay Technology in HTS

26. Requirements for screening assays -> High ThroughputScreening Assay • High sensitivity of assay (single molecule detection) • High speed of assay (automation) • Minimization of assay (microtiter plate assay) • Low background signal • Clear message (best: Yes/No answer) • Low complexity of assay (specific interaction) • Reproducibility • Fast data processing of results • Acceptable costs !!!

27. Screening for novel compounds Laboratory Automation Beginning in late 1980s -> - microtiter plate assays - liquid handling automats - plate readers Speeded up by -> miniaturization and parallelization of assays (96-1536 well plates) Assays can be run: -> semi manual (batch mode) small differences in timing of the steps -> additional assay noise (time jitter) -> continuous (robotic integration of processing automats) since cycle time are constant -> easier to identify systematic trends and errors more complex logistic

28. Screening for novel compounds Laboratory Automation – Assay automation

29. Screening for novel compounds Laboratory Automation 1 cycle -> 230s Overnight run (16h): -> 250 microtiter plates -> 384 000 tests

30. Screening for novel compounds From target selection to confirmed hits Identification of drug targets Choice of assay technology: -> expertise -> compatibility with existing HTS hardware -> sensitivity -> profile of drug -> susceptibility to artifacts -> unspecific interference -> cost of assay -> reproducibility Primary test: -> final assay concentration cannot be controlled -> signal scattering -> liquid handling errors -> signal scattering -> false negative (cannot be excluded) -> active compounds not detected (5-15%) -> false positive (-> cross-contamination, assay noise) -> identified by repetition -> controls are very important!!!!

31. Screening for novel compounds From target selection to confirmed hits Screening inhibitors of a serine protease -> Completed in 2 weeks 9 robots testing 170 000 a day A -> compounds with 20% And more inhibition were Considered B -> activity in re-testing Compared to primary test Degree of reproducibility -> false positive C -> evaluation -> conformation rate correlates positive with activity

32. Screening for novel compounds Specific <-> unspecific hits (assay artifacts) In HTS -> small number of lead candidates -> large number of unreproducible hits (false positives) -> compounds acting unspecific Unspecific acting -> same effect on assay as the desired drug !!! Interference increases -> complexity of assay !!!! -> cell-based assays -> reference assays -> probing for specificity and selectivity (challenging)

33. Screening for novel compounds Specific <-> unspecific hits (assay artifacts) Discrimination between Target molecule and unspecific Acting molecules. -> cell-based assay Inhibitor for G-protein coupled Receptor (GPCR) Activity monitored by release of Ca2+ -> Ca2+ concentration was visualized by Ca2+ sensitive photoprotein -> connected to stimulation of ATP Library: 1 000 000 compounds -> 10 000 hits (too many) ATP signal as reference to find unspecific hits Specific hits -> enhancing response to ATP -> 656 hits

34. Screening for novel compounds Data analysis and screening results Data analysis combines -> biological activity + chemoinformatic tools evaluate + interpreting experimental observations Grouping active compounds into structural classes (clusters) -> enhances resolution looking at scattered data

35. Screening for novel compounds Data analysis and screening results Chemoinformatics in HTS: -> Elimination of unspecific compounds from hit sets -> detection of false negative + borderline activity • Search library for: • Overall similarity • shared substructure • known bio-isosteric replacements Computer tool: -> identify molecular fragment contributing to biological activity Possible to extract information that is not detectable by cluster methods

36. Screening for novel compounds !!! Experimental testing remains the major route for lead discovery !!! Prerequisite for success: Convert knowledge of mechanism + molecular recognition principle -> robust + sensitive assay -> HTS should deliver more than one candidate !!!

37. End 1st lecture

38. Detection Methods in HTS: • Spectroscopy • Mass Spectrometry • Chromatography • Calorimetry • X-ray diffraction • Surface plasmon resonance • Microscopy • Radioactive methods

39. Spectroscopy in HTS: • Fluorescence Spectroscopy • Total internal reflection fluorescence (TIRF) • Nuclear magnetic resonance (NMR) • Absorption and luminescence sp. • Circular dicroism sp. (CD) • Fourier transformed infrared sp. (FTIR) • ATR-FTIR • Light scattering

40. Fluorescence Spectroscopy in HTS: • Fluorometric Imaging Plate Reader • Fluorescence polarization spectroscopy (FP) • Time-resolved fluorescence spectroscopy (TRF) • Fluorescence resonance energy transfer spectroscopy (FRET) • Fluorescence correlation spectroscopy (FCS) • Fluorescence correlation microscopy (FCM) • Confocal fluorescence coincidence analysis (CFCA)

41. Fluorometric Imaging Plate Reader 384 samplescan be measured simultaneously in using a FLIPR (Fluorometric Imaging Plate Reader). The fluorescent species measured is a sensitive dye. Using 384-well microplates with a 5-min turnaround time, nearly 40,000 samples can be screened in an 8-h period.

42. Fluorescence polarisation (FP): FP (Fluorescence polarization) can measure the binding of a small molecule to a much larger protein. According to FP theory, unbound fluorescent tracers will exhibit lower polarization, because the unbound molecules tumble more rapidly than bound tracers.

43. Fluorescence polarisation (FP): • Fluorescence polarization (FP) is a useful analytical technique that can measure the extent to which a fluorescent tracer (typically a small molecule) binds to a large molecule, such as an enzyme, nucleic acid, or antibody. • In general, a molecule's rate of tumbling (more formally known as the rotational relaxation time) is directly proportional to its molecular volume at constant temperature and viscosity. Small molecules tumble rapidly, large molecules tumble slowly. If a small fluorescent molecule is bound to a large molecule, however, then it will tumble slowly. • By measuring the extent of fluorescence polarization, this method can determine binding equilibrium and the competition for binding at a site. For example, suppose a company wants to screen for compounds that are enzyme inhibitors and that bind strongly to an active site. The FP assay would use a fluorescent marker that also binds to that enzyme's active site. Stronger inhibitors would compete more successfully for the binding sites, thus releasing more of the fluorescent marker into the unbound state. These samples would show a greater degree of random depolarization.

44. Applications of FP: • well suited for assays that rely on small ligands binding to large biomolecules, including enzyme assays (kinase assays, protease assays) and immunoassays (pesticides, metal ions)… • Used for diagnostic assays (disease detection), drug discovery, signal transduction discovery, single nucleotide polymorphism genotyping (identification of genetic variations in the human genome)…

45. Time-resolved fluorescence spectroscopy (TRF)

46. Time-resolved fluorescence (TRF): • Time-resolved fluorescence (TRF) is closely related to fluorescence intensity techniques. In TRF, the detector is gated for a short period of time (e.g., 10 ns) so that the initial burst of fluorescence, including most of the background fluorescence, is not measured. After the gating period, the longer lasting fluorescence in the sample is measured. TRF techniques can be used to substantially enhance sensitivity levels. • This technique, of course, requires that the signal of interest must correspond to a compound with a longer fluorescent lifetime. The long-lived fluorescent compounds that make TRF feasible are e.g. rare earth lanthanides. Because of their unfilled 4fn orbitals, lanthanides have interesting and useful photochemical, electrical, and magnetic properties, including long fluorescent lifetimes. • TRF has been further enhanced with the development of a class of cage compounds called cryptates. Cryptates are macropolycyclic compounds that can serve as cages, trapping an ion and protecting it from solvent. The cryptate cage enhances fluorescence by acting as an antenna for the trapped lanthanide ion (i.e., absorbing excitation light and transferring the energy to the ion) and by protecting it from quenching by water. These compounds were discovered by Jean Marie Lehn, who received the 1987 Nobel Prize in Chemistry for this work. • Using Eu cryptate, Eu(K), in conjunction with a second scientific phenomenon, scientists have now developed a homogeneous technique that can be used in HTS systems. It uses the principle of fluorescence resonance energy transfer (FRET). This technique is also called time-resolved, fluorescence resonance energy transfer, or TR-FRET.

47. Fluorescence resonance energy transfer spectroscopy (FRET) : In HTRF (Homogeneous Time-Resolved Fluorescence),fluorescence depends on bringing the donor and acceptor fluorophores together. In an HTRF assay, the transfer of resonance energy between a donor and acceptor fluorophore provides a way to measure ligand binding to a biological target. Two conditions must pertain for an effective FRET assay 1) The donor fluorescence must be significantly quenched 2) The starting material and product need to differ significantly in extent of quenching (Forster radius ca. 50 A)

48. Fluorescence resonance energy transfer spectroscopy (FRET) • FRET is a technique which is based on the principle of time-resoved fluorescence (TRF). • FRET refers to the nonradiativeenergy transfer from a donor fluorophore to an acceptor fluorophore. The efficiency of energy transfer is a function of the distance (1/d<SUP6< sup>) between the donor and acceptor. In TRF, the donor is (Eu)K (molecular weight ~1000), and the acceptor is XL665 (a modified allophycocyanine, a phycobiliprotein from red algae, molecular weight ~105,000). This donor- acceptor pair has a 50% energy transfer efficiency at a distance of 9 nm and a 75% energy transfer efficiency at 7.5 nm. The transfer distance is long enough to be compatible with biomolecular interactions of interest, yet short enough to minimize nonspecific energy transfer. • The most popular FRET pair for biological use is a cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) pair. Both are color variants of green fluorescent protein (GFP). While labeling with organic fluorescent dyes requires troublesome processes of purification, chemical modification, and intracellular injection of a host protein, GFP variants can be easily attached to a host protein by genetic engineering.

49. Fluorescence resonance energy transfer spectroscopy (FRET) • A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor or to photobleaching. To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. This technique uses a bioluminescent luciferase (typically the luciferase from Renillareniformis) rather than CFP to produce an initial photon emission compatible with YFP. • FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors.

50. Application of TRF and FRET: • based on the principle of dissociative fluorescence enhancement: immunoassays, DNA hybridization assays, cytotoxicity assays, protein-protein binding, receptor binding, enzyme assays,… • Various reagents pre-labeled with TRF donors and acceptors, including epitope tag antibodies, streptavidin, biotin, and several antispecies antibodies, are available and can be adapted to many assays.