High throughput screenig
- 1. HIGH THROUGHPUT SCREENING MAHAMMAD SHAKHEEL B PHARMACOLOGY DEPARTMENT
- 2. •HTS IS A METHOD FOR SCIENTIFIC EXPERIMENTATION ESPECIALLY USED IN DRUG DISCOVERY AND RELEVANT TO THE FIELDS OF BIOLOGY AND CHEMISTRY. •DATA PROCESSING, AND CONTROL SOFTWARE, LIQUID HANDLING DEVICES, SENSITIVE DETECTORS- HTS ALLOWS A RESEARCHER TO QUICKLY CONDUCT CHEMICAL, GENETIC AND PHARMACOLOGICAL TESTS. •THE RESULTS OF THESE EXPERIMENTS PROVIDE FOR DRUG DESIGN AND ROLE OF BIOCHEMICAL PROCESS IN BIOLOGY. High throughput screening
- 3. Assays for HTS and validation play a key role in drug discovery and development. Currently, most HTS assays are rely on the use of fluorescent and radioactive labels to messure bichemical reactions or monitor cellular responses. Lables have adverse effect on binding interaction being investigated leading to false conclusion about binding properties of analyte Lables also introduce additional complexity and assay develoment time to the procees.
- 4. Commonly used terms in drug discovery High throughput screen: an optimised, miniaturised assay format that enables the testing of > 100,000 chemically diverse compounds per day. Assay: a test system in which biological activity can be detected Hit: a molecule with confirmed concentration-dependent activity in a screen, and known chemical structure. The output of most screens
- 5. Progressible hit: a representative of a compound series with activity via acceptable mechanism of action and some limited structure-activity relationship information Lead: a compound with potential (as measured by potency, selectivity, physico-chemical properties, absence of toxicity or novelty) to progress to a full drug development programme Pharmacophore: minimal structure with essential features for activity
- 6. Drug discovery Initial characterisation trials Regulatory approval sought to start trials in humans Pre-clinical Clinical trials Phases I, II, III Submission of marketing/manufacturing authorisation application to regulatory authorities or refuse) licences Product goes on sale Post-marketing surveillance Regulatory authorities review information and grant ( Library of compounds In vitro screening: human/animal receptor/enzyme assay; reporter system Hits/lead Biochemical, tissue or animal model of function lead Animal model of therapeutic target ADME, formulation, acute toxicology The life history of a successful drug
- 7. High throughput screening for drug discovery FACT 1: recent understanding of disease mechanisms has dramatically increased no. of protein targets for new drug treatment FACT 2: new technologies have increased the no. of drugs that can be tested for activity at these targets. high throughput screening (HTS) is 1° tool for early-stage drug discovery
- 8. HTS is process by which large nos. of compounds are rapidly tested for their ability to modify the properties of a selected biological target. Goal is to identify ‘hits’ or ‘leads’ - affect target in desired manner - active at fairly low concs (∴ more likely to show specificity) - new structure The greater the no. and diversity of compounds screened, the more successful screen is likely to be. HTS = 50,000-100,000 cpds screened per day!!!
- 9. Goals and limitations of HTS Aim of screening is to find progressible hits, not to discover the lead molecule itself The majority of drug targets are a) G-protein coupled 7 TM receptors b) nuclear receptors c) ion channels d) enzymes progressible hit targeted synthetic design Lead
- 10. Take top 100 drugs - 18 bind to GPCR - 10 bind to nuclear receptors - 16 bind to ion channels - most of remainder inhibit enzymes Knowledge gained from one drug target can be transferred to related targets. e.g molecular technology required to work with 1 GPCR is useful for other GPCRs, including cloning and expression systems and info on structure and ligands.
- 11. Implementation of HTS Need 4 elements: 1) suitable libraries of compounds Source of chemicals for screen: - in-house collection (5x105 - 106 ) of diverse samples. - supplement by acquisitions from specialist companies -combinatorial chemistry allows synthesis of large no of diverse molecules. 2) assay method configured for automation Assay requirements: a) pharmacology of the target should not be altered by the molecular manipulations
- 12. b) cost of assay development and reagents low c) easy to use and suitable for automation and miniaturisation. Use multi-well plates: 96, 192, 384, 864, 1536 and assay requiring few manipulations, no plate-o-plate transfers or washing steps d) robust signal-to-noise ratio. Hit defined as activity above a certain threshold e.g. Ki < 5 nM Emax >30% increase over basal e) ideally be non-radioactive Often express target genes in appropriate host systems e.g. bacterial, yeast, viral, invertebrate and mammalian cells.
- 13. a) Radioligand binding assays • Measures affinity of library compounds for target. • Need high affinity radioligand that binds to site of interest and cells transfected with target site. • measure competitive displacement of radioligand from target site • Specificity can be assessed by including other possible targets in screen relative affinity for multiple sites PROBLEM: hard to miniaturise radioactive assays (counting takes too long). Alternative is to use fluorescence techniques.
- 14. b) Cell-based fluorescence and radiotracer assays Useful for measuring ion-channel function e.g.measure movement of Ca2+ in a fluorescent-imaging plate-reader (FLIPR) • cells are loaded with the fluorescent Ca2+ indicator Fluo-3 • depolarisation with high KCl activates Ca2+ channels and allows Ca2+ entry Ca2+ Ca2+ -60 mV fluorescence signal 488 nm Ca2+ Ca2+ -30 mV increased fluorescence 488 nm (a) rest (b) with KCl
- 15. c) melanophore assays Melanophores = pigmented cells derived from neural crest. Prepare immortalised melanophores from Xenopus laevis Gi melanophores transfection CMV Hß2-AR Plasmid vector AGGREGATED MELANOSOMES melatonin DISPERSED MELANOSOMES light Gs, Gq ß agonist ß antagonist
- 16. d) Reporter gene assays Rather than measure the immediate cellular response, it may be easier to measure the subsequent transcriptional change isoprenaline binding to ß-adrenceptors cAMP PKA activation and translocation to nucleus phosphorylation of transcription factor CREB that recognises cAMP response elements (CREs) expression of reporter gene whose transcription is driven by an enhancer containing CREs Measure reporter gene product in HTS format
- 17. • Examples of reporter genes: ß-galactosidase; luciferase; alkaline phosphatase; green fluorescent protein. • Useful for measuring responses from Gi, Gs or Gq-coupled receptors
- 18. All screens have danger of false negatives and false positives Not such a problem waste time and resources HTS is less useful for evaluating - bioavailability - pharmackinetics - toxicity - absolute specificity 3) robotics workstation • Robots handle assays in multi-well formats. - sample dilutions - sample dispensing - plate washing
- 19. Hard to automate cell lysis or permeabilisation steps (necessary for many 2nd messenger responses). • Full automation allows 24 h continuous operation without requiring shift work. • More efficient and economical. 4) computerised data handling system •A great deal of data is generated. Must be accurate and reproducible. Need good computerised data handling systems.
- 20. Which strategy is best for hit identification? When a target is identified, a decision has to be made about which chemicals to screen, in order to identify potential lead compounds. Random screening All possible drug molecules screened against target. Estimated no. of possible drug molecules is ± 1040 !!! This is simply not possible.
- 21. Focussed screening A limited number of compounds are pre-selected for screening. Has proved successful as a hit generation strategy. Useful when 3D structure of target is known (e.g. crystal structure of a receptor) - use computer modelling to predict optimal structure to interact with target - use known ligand to construct 3D pharmacophore In either case, select compounds from library or design new compounds and screen. Focussed screening will find novel hits BUT the required information may not be available.
- 22. Choice of expression system for GPCRs • Ligand binding and transduction properties are effected by cell type, receptor density, and presence of signal transductional elements ADVANTAGES Yeast-based systems e.g. Saccharomyces cerevisiae (also used to express protein tyrosine kinases, peptide hormones and functional ion channels) - grow rapidly - easy and cheap - readily amenable to genetic manipulation - tolerant to solvents used to dissolve drugs e.g.DMSO, methanol Bacterial cells e.g. E.coli - grow rapidly - easy and cheap DISADVANTAGES - May not get functional coupling of transfected GPCRs to yeast G-proteins. Overcome this by coupling GPCRs to yeast pheromone response pathway agonists promote cell growth or activity of reporter gene construct - may not get proper post-tranlational modification e.g. many GPCRs are not glycosylated in yeast, or cell surface expression. - Low expression levels - Some post-translational modifications not performed - no endogenous G-proteins
- 23. ADVANTAGES Baculovirus/insect cell system e.g. Sf9 cells - high levels of expression of functional protein - ligand binding properties similar to native receptor - can co-express receptor and mammalian G-proteins - ideal for structural analysis Mammalian cells e.g. CHO, HEK - most authentic background for expression of GPCRs • protein synthesis • membrane insertion • post-translational modification • lipid composition of membranes - receptors usually functional DISADVANTAGES - production of permanent cell lines may be a difficult and lengthy process - can be expensive - receptor promiscuity (different G-proteins activated by 1 receptor multiple signals) - no. of suitable G-proteins may be limited - GPCR might not be glycosylated - GPCR is occasionally inactive
- 24. Yeast-based screen and selection for identification of agonists for a human orphan GPCR Yeast ligand Yeast receptor Yeast G protein arrest human ligand human receptor human G protein growth (a) In yeast, normal GPCR-initiated pathway results in cell cycle arrest in response to agonist (b) Substitution of human orphan GPCR and human Gα protein yields a strain that only grows if receptor is stimulated Spot individual cpds onto lawn of modified yeast. Detect agonists by growth of yeast on the plate around the site of cpd application
- 25. orphan receptor peptide growth peptide expression library peptide ligand (c) alternatively, introduce a random peptide expression library; cells will only grow if the peptide stimulates the receptor
- 26. REFERENCE Short protocols in Pharmacology and Drug discovery edited by Enna S J, et al. Drug discovery and evaluation Pharmacological assy by Vogel H G and Vogel H G. Modern methods of Drug Discovery by Hillisch, A and Hilgenfeld.
- 27. THANK YOU