hit identification.pptx

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1. Seminar presentation on Assay Development for hit Identification Presented by: Soheila Ahanjan M.Pharm 2 nd semester Department of Pharmacology SPER, JAMIA HAMDARD 1

2. Content Introduction Assay Development Factors important in Assay Development High Throughput Screening Biochemical assays Cell based assays Automation and Robotics in HTS Applications References 2

3. Introduction Hit identification is the most critical step to identify compounds able to interact with the fully validated target. There are different strategies available to identify good hits, devoted to maximizing the number of initial hits and hit series. At the same time, the strategy must ensure it reduces the amount of potential false positives, thereby reducing the risk of identifying a series of compounds that are suboptimal for development, spending resources on the wrong compounds. A successful hit identification relies on the use of a high-quality, diverse library of potential compounds to be screened, but also capitalizing on other complementary techniques, such as virtual screening and fragment-based screening. 3

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5. Assay Development Assays are investigative procedures that qualitatively assess a compound or examine a compound’s effects on identified molecular, cellular, or biochemical targets. In the following stage of drug development, biological assays and compound screening assays are created. These assays are used to identify compounds that have a desired activity at the drug target. These compounds are referred to as “hit” molecules. During the initial phase of hit compound identification, termed high throughput screening (HTS), a compound library that contains many potential hit molecules is tested to identify any compounds with the desired activity towards the target. 5

6. Cont. Further assays are required to retest the hit molecule’s activity at the target. Finally, cell-based assays are used to examine a drug’s toxicity, safety profile, and efficacy. Every drug that is developed undergoes a unique series of assays that are specifically designed and organized for the drug target and compound in question. This process is termed assay development. 6

7. Factors important in Assay Development FACTOR IMPORTANCE IN ASSAY DEVELOPMENT Relevance Research should be conducted to examine the ability of the assay to: 1.Predict the specific disease state. 2.Identify compounds that exhibit an appropriate mechanism of action and strength. Reproducibility In a compound screening environment, an assay must be reproducible. This means that feasible reproducibility exists across assay plates, screen days and the full duration of the specific drug discovery program. Quality Z’-factor is calculated to measure the power or quality of an HTS assay. The signal window and variance of negative and positive signals is used in this calculation. The Z’-factor must be >0.4 to be considered acceptable for use, with some researchers preferring a Z’-factor of >0.6. Interference Assays must be designed that consider the effects of compounds found in the assay, such as the solvents used . 7

8. High Throughput Screening HTS is a process to accelerate drug discovery, which involves a brute force approach where tens of thousands of compounds (Compound libraries) are tested against a particular target daily. This method is very valuable to early drug discovery. Compounds are being tested using a quantitative bioassay via the use of automation, miniaturized assays, micro fluidic chips, sub nano litre dispensing, fluorescence, large-scale data analysis. High-throughput screening methods are also used to characterize metabolic, pharmacokinetic and toxicological data about new drugs. This HIT compound can be generated into LEAD compound. 8

9. High throughput screening (HTS) involves the screening of the entire compound library directly against the drug target or in a more complex assay system, such as a cell-based assay, whose activity is dependent upon the target but which would then also require secondary assays to confirm the site of action of compounds. Basically HTS is a process of screening and assaying large number of biological modulators and effectors against selected and specific targets. Principle of High Throughput Screening: 9

10. Methods which are commonly followed are: 1) Target selection: Currently there are about 500 targets being used by companies. cell membranes receptors, mostly G-protein coupled receptors make up the largest group (45% of the total), Enzymes make up the next largest group (28%), followed by hormones (11%), unknowns (7%), ion-channels (5%), nuclear receptors (2%), and finally DNA (2%). 10

11. Cont. 2) HTS library to be screened: i) they usually consist of microtiter plates with frozen or dried samples of compounds to be screened. ii) Initially the assays were carried out in 96-well plates but with advancement now there are also 1586-well plates available. iii) Typical HTS programs have potentials to screening up to 10000 compounds per day, while some laboratories with Ultra High Throughput Screening (UHTS) can perform100,000 assays per day. 11

12. Cont. 3) Assay design: Assays mainly divided into biochemical and cell based assays. Biochemical assay are further divided into two homogenous and heterogenous assays. 3.1) Homogenous assay: i) Measurement are based on the distinct physical/chemical properties of analyte, or interaction between analyte and surrounding environment. ii) It is a single step process; reagent may be added at single stage or in multiple steps. iii) It only involves usual steps like fluid addition, incubation and reading. 12

13. Cont. It can be coupled with different detection technique fluorescence, radiometric for HTS. Advantage: i) Simplicity(Mix and read) ii) Reduction of cost and robotic complexity. 3.2) Heterogenous assay: i) Heterogeneous assays involves additional steps like filtration centrifugation. that separates component(s) to be measured from the rest of component which may interfere in assay. ii) contributes to the high signal to background ratio. 13

14. Biochemical assays: i) Biochemical assays are receptor, protein or enzyme based assays uses the particular target in a purified form. ii) Biochemical assays are most frequently carried out using scintillation proximity assay(SPA), radiometric, colorimetric fluorescence detection techniques. iii) Scintillation Proximity Assay is a technology whereby binding reactions can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. 14

15. Some techniques of biochemical assays are: Fluorescence resonance energy transfer(FRET): a) In this process, donor fluorophore absorbs the energy from incident light and transfer this energy to nearby acceptor molecule. b) One frequent pair fluorophores is a cyan fluorescent protein (CFP)- yellow fluorescent protein(YFP) pair, spectrally distinct variants of green fluorescent protein. Graph of FRET (b) Jablonski diagram illustrating the FRET process. 15

16. ii) Fluorescence polarization: a) When fluorophore is irradiated with light, it gets exited and if remain steady throughout excitation state, it emits light in same polarized plane. b) While if it rotate and tumbles during excitation state, it emits light in different plane(depolarized). c) Larger molecule shows little movement while small molecule rotates quickly and gives high and low polarization value respectively FP is widely used in HTS. 16

17. Cell Based Assays Cell-based assays for HTS can be classified under following classes: 1) Second Messenger Assay: a) It monitors signal transduction from activated cell-surface receptors. b) Second messenger assays typically measure fast, transient fluorescent signals that occur in matter of seconds or milliseconds. c) Many fluorescent molecules are known to respond to changes in intracellular Calcium ion concentration, membrane potential and various other parameters, hence they are used in development second messenger assays for receptor stimulation and ion channel activation. 17

18. Cont. 2) Reporter gene assays: a) It monitors cellular responses at transcription/translation level. b) It indicates the presence or absence of a gene product that in turn reflects changes in signal transduction pathway. c) The quantification of the reporter is usually carried out by biochemical methods viz by measuring the enzymatic activity. 3) Cell proliferation assays: a) It monitors overall growth/no growth responses of the cell to external stimuli. b) Quick and easy to be employed for automation. 18

19. 4) Reagents in HTS: In any chemical synthesis or testing and screening, reagents play a major role, HTS is no exception to this. Examples of reagents used are: i ) Aptamers ii) Enzymes (e.q: Tyrosine kinase) iii) Biotinylated Deoxy uridine Triphosphate iv) Streptavidin- allophycocyanin v) Dimethyl sulfoxide (DMSO) 19

20. Automation and Robotics in HTS: The union of robotics and HTS has been important to achieve the desired screening rates, as well as relieving scientific staff from tedious work. Problems associated with screening robotics have included long design and implementation time, long manual to automated method transfer time, non-stable robotic operation, and limited error recovery abilities. 20

21. 21 The presently used robot-centric HTS systems have a central robot with a gripper that can pick and place microplates around a platform. They typically process between 40 and 100 microplates in a single run (the duration of the run depends on the assay type).

22. Applications Selection of compounds from a vast number synthesized by combinatorial chemistry and other methods. Efficient tool in studying biomolecular interactions and pathways. Highly efficient, fast, accurate and dependable in compound screening. Used in DNA sequencing. Useful in toxicology. Used to study drug-drug interactions. Useful in genotoxicity assays. 22

23. References https://www.irbm.com/drug-discovery/hit-identification/#:~:text=Hit%20identification%20is%20the%20most,initial%20hits%20and%20hit%20series. https://www.technologynetworks.com/drug-discovery/articles/assay-development-329953 Hughes JP, et.al . Principles of early drug Discovery. British Journal of Pharmacology 2011; (162) :1239–1249. Armstrong, J.W. A review of high-throughput screening approaches for drug discovery. Am. Biotechnol . Lab 2018; 17: 26-28 23