HTS

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A Seminar on High Throughput Screening Presented By:- Prerna Chauhan

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INTRODUCTION High throughput screening is a method for scientific experimentation. Especially used in drug discovery and relevant to the fields of biology and chemistry. HTS allows to conduct millions of biochemical, genetic or pharmacological tests by using specific programmes, data processing, control software, liquid handling devices and s ensitive detectors.

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HTS helps in rapid identification of active compounds, antibodies or genes which modulate a particular biomolecular pathway. Result of these experiments provide starting points for drug design and for understanding the interactions or role of a particular biochemical process in biology .

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The last two decades have seen innovations in the technology that have helped to evolve manual Low speed screening into an  automated, microprocessor controlled programmed process called “High Throughput Screening (H.T.S)”. HTS has helped to speed up LTS and now over 50,000-100,000 compounds can be scanned per week , against the validated biological target. valuable compounds can now be selected rapidly from a large number of samples with minimal human and cost involvement. HISTORY OF HTS:-

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STEPS OF HIGH THROUGHPUT SCREENING:-

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Steps involved in Drug Development research

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Discovery of the new Lead compounds for Novel therapeutic targets is a multi-step process involving: Drug design Synthesis Pharmacological screening As the first step towards the drug development, the biological targets needs to be identified. In the next step, the potential compounds are screened against the identified target.

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INSTRUMENTATION AUTOMATION SYSTEMS:- Automation is an imp. element in HTS’s usefulness. An integrated programmed system consisting of one or more programming devices, transport assay microplates from station to station for sample and reagent addition, mixing, incubation and finally readout or detection. HTS can test upto 100000 compounds per day.

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ASSAY PLATE PREPARATION:- Testing vessel of HTS: Microtitre Plate A small container, made of plastic and having a grid of small open divots, called wells.

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Each microplate consists of 96 wells. Original microplates having 8×12.9 spaced wells. Modern microplates have either 384, 1536 or 3456 wells, i.e. multiple of 96. Large no. of wells contains experimental matter i.e. aqueous solution of DMSO and some other contains chemical compounds i.e. different for each well across plates.

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A screening facility holds a library of stock plates whose contents are carefully catalogued. These stock plates, themselves are not directly used in exp., instead separate assay plates are created as needed. An assay plate is a copy of stock plate. Created by pipetteing a small amount of liquid from the well of a stock plate to the corresponding wells of a completely empty plate.

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REACTION OBSERVATION Preparation of assay- Each plate is filled with experimental biological entity. Allows biological matter to absorb, bind to, or react with compounds in wells. Measurements are taken across all the plate’s wells.

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A specialized automated analysis machine can run a no. of experiments on the wells such as shining polarized light on them and measuring reflectivity which can be an indication of protein binding. In this case, the machine outputs the result of each experiment as a grid of numeric values with each no. mapping to the value obtained from a single well.

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Depending on the result of this first assay, followup assay can be performed with in the same screen by “cherry picking” liquid from the source wells. It gave result into new assay plates and then re running the experiment, to collect the further data on this narrowed set, confirming and refining obeservations.

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TECHNIQUES OF HTS AND ITS APPLICATIONS:- In HTS, the intraction of ligands with the biological compartment is elucidated by luminescence based binding assays. In this manner, several thousands of compounds from chemical library can be assessed for their binding .

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Various techniques are- IN VITRO ASSAYS:- FLUOROSCENCE ANISOTROPY FLUOROSCENCE CORRELATION SPECTROSCOPY FLUOROSCENCE RESONANCE ENERGY TRANSFER CELL BASED ASSAYS TO STUDY INTRACELLULAR EVENTS SCINTILLATION METHODS IN DRUG SCREENING IN VIVO IMAGING OF DRUG ACTION WHOLE BODY IMAGING TECHNIQUES(SPECT) VIRTUAL SCREENING

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IN VITRO ASSAYS: FLUOROSCENCE ANISOTROPY- Also called FLUOROSCENCE POLARIZATION. This technique uses plane polarized light to excute the fluoroprobe. Low polarization has been observed when ligand is free in solution. Binding restricts the rotational mobility of fluoroprobe, thereby, a higher polarization is exhibited. Polarization value of a molecule is proportional to rotational relaxtation time depends on molecular volume.

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EXAMPLE- Specific binding of fluoroscin-trp25-exendin-4 to human glucagon- like peptide 1 receptor was reported using FA. Applications- Suitable to detect receptor–ligand binding reactions.

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FLUOROSCENCE CORRELATION SPECTROSCOPY- In this, a narrow focused laser beam excites the fluoroprobe in small volume. Each single molecule diffusing through illuminated area produces quanta of fluoroscent light while they are exhibiting brownian movement.

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These individual bursts are recorded as a function of time by sensitive detector. Using software integration, it gives the details of concentration, diffusion time and intensity of all molecules.

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ADVANTAGE- Very less amount of material is used. Relatively short measurement times. APPLICATIONS - Used to study the protein –ligand intractions.

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FLUOROSCENCE RESONANCE ENERGY TRANSFER- FRET occurs when two different chromophores interact via dipole –dipole Mechanism, where the excited chromophore transfers its excitation energy to a closely located acceptor chromophore.

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APPLICATIONS- Rate of monoclonal antibodies (tagged with Cy5) binding with interleukin 1 receptor(tagged with europium chelate), to form a stable complex, has been monitored by FRET .

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CELL BASED ASSAYS TO STUDY INTRACELLULAR EVENTS- Fluoroscent indicators of calcium are developed to study changes in Intracellular calcium levels using fluoroscence microscopy or fluoroscence spectroscopy. These fluoroscent probes are structurally related to calcium chelators like EGTA and BAPTA.

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it shows a rapid spectral change on binding of calcium, a feature that make them ideal for kinetic calcium measurements. Newly developed Fluorometry Imaging Plate Reader(FLIPR) is capable of reading fluoroscent calcium signals all 96 wells simultaneously .

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SCINTILLATION PROXIMITY ASSAY- This system uses the principle that an antibody or a receptor molecule, which is bind to a bead , emits light when beta emission from an isotopeoccurs in close proximity i.e. when a radiolabled ligand binds to a bead with receptor or antibody. The basic technology of SPA is based on the fluoroscent signal produced by a scintillant – dyed polystyreneor polyvinyl toluene microspheres that could be excited by the proximity of a radiolabled molecule.

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Scintillant dyed microspheres are in size range of < 2 µm in diameter. APPLICATIONS- Used in evaluation of kinetics of protein kinase inhibitors, To evaluate neurotransmitter transporter inhibitors, Evaluation of reverse transcriptase inhibitors etc.

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IN VIVO IMAGING OF DRUG ACTION(NEAR INFRARED IMAGING)- It is a newly added method to evaluate the drug action and quantification without killing the animal. Fundamental of this technique is using molecules capable of emitting light in higher wavelength near IR region, thereby, it gains the capability of crossing biological membranes and to be detected by sensitive camera turned for near IR region. For this application, an exogenous contrast agent like indocyanine green is used.

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APPLICATIONS- Utilized in vascular mapping, tissue perfusion studies, Imaging tumours, Inflammation, Atherosclerosis Cell death etc.

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WHOLE BODY IMAGING TECHNIQUE SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY:- Highly sensitive nuclear medicine tomographic imaging technique Using gamma rays. SPECT imaging involves the administration of radiotracers into the biological system and the radiations emitted from the radiotracer in the body are captured by the gamma cameras with sodium iodide crystal detector

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and photomultiplier tube in which the radiations are converted into scintillation photons and amplified and the images are reconstructed by computer in such away that the image of organ of interest is obtained. APPLICATIONS- In study of involvement of drug efflux transporters like multidrug resistance associated protein, lung resistance protein, as a contributing factor for MDR in cancer therapy in patients.

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VIRTUAL SCREENING:- It is totally newer approach using advanced computer technology to screen Newer Compounds based on virtual coordinates of receptor and ligands. Computer –aided molecular design(CAMD) approach involves computational analysis of large data set in order to highlight those compounds most likely to be active in the actual assay, so that a focused subset of compounds can be selected. Virtual screening needs the 3-D molecular structure of the receptor along with the 3-D structure of ligand to perform docking.

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CONCLUSION:- HTS has tremendous impact on drug discovery. Luminescence based techniques and newer developments using radioactive compounds in SPA have given a new direction for the drug discovery process. Most of the instruments are now affordable for lab working with a considerable budget. This technology in every field is expected to increase to many folds and would be creating a method through which more and more safe drugs would be discovered for almost all the diseases.

REFERENCES:

REFERENCES 1.Gupta S.K, ”Drugs screening methods”2 nd edition.p.no-17-33. 2.http:\\www.bmglabtech.com\application-notes\HTS\assay category. 3.Journal of Biomolecular Screening vol-4,no.2,1999.