HIGH THROUGHPUT SCREENING.pptx

HIGH THROUGHPUT SCREENING

1. ADVANCED SCREENING TECHNOLOGY FOR NATURAL PRODUCTS

2. CONTENTS INTRODUCTION DRUG DISCOVERY PROCESS HIGH THROUGHPUT SCREENING APPLICATIONS CONCLUSION

3. INTRODUCTION HTS is basically a process of screening and assaying huge number of biological modulators and effectors against selected and specific targets. The principle and methods of HTS find their applications for screening of combinatorial chemistry, genomics, protein and peptide libraries. The main goal of this technique is to hasten the drug discovery process by screening large compound libraries.

4. OVERVIEW OF DRUG DISCOVERY PROCESS OF NATURAL PRODUCTS

5. HIGH THROUGHPUT SCREENING It is a process by which very large number of compounds from variety of sources{synthetic collections, natural products extracts, combinatorial chemistry libraries} are tested against biological targets. it is presently the most important & high procreative power for useful & ultimate discovery of a variety of newer lead chemical entities in pharmaceutical industry.

6. HTS indeed makes use of extremely specific miniaturized assay formats having the following salient features: Uses microstate plates capable of handling 384 sample variants that may be assayed most conveniently at < 50 microliter total assay volume per run effectively. Fully automated device and one may carry out the assay of hundreds of thousands of sample against each biological target of interest.

7. Successful HTS program integrate several activities including: Target identification Reagent preparation Assay development Data analysis & management High throughput library screening

8. TARGET IDENTIFIATION One of the first activities in developing a HTS assay is selecting the target. Of these 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%). The target must be part of some regulatory pathway in the cell & should be sensitive to some disease state not to be expressed all the time.

9. REAGENT PREPARATION In any chemical synthesis or testing and screening, reagents play a major role, HTS is no exception to this. Reagents must be characterized and optimized before use. Aptamers , nucleic acids that bind to other molecules high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small molecule ligands to protein targets. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labelling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Enzymes are often used as regents in HTS, an example Tyrosine Kinase was used to find its inhibitors. In this care must be taken that in reagent preparation there should not be any

10. contamination with other kinases, phosphatases, and peptidases which may compete with Tyrosine Kinase to give false results. Other than kinase enzymes, generic reagents like biotinylated Deoxyuridine Triphosphate, Streptavidine-allophycocyanine , and Streptavidine -europium were used developed for determing the activity of HIV-Reverse Transcriptase. 3 Dimethyl sulfoxide (DMSO) is another widely used reagent as it is preferred vehicle for compound /sample delivery. The important point to remember during the use of DMSO is that its tolerance should be determined early during assay development stage so as to carry out further optimization during the screening stage.

11. HTS ASSAY

12. BIOCHEMICAL ASSAY

13. TECHNIQUES OF BIOCHEMICAL ASSAY Fluorescence resonance energy transfer (FRET): It is the non-radioactive transfer of energy between appropriate energy donor and acceptor molecules. Fluorescence polarization (FP): Its measurements allow one to measure changes in the rotational diffusion coefficient of small labelled probes upon binding to larger molecules. Homogeneous time resolved fluorescence (HTRF): Fluorescence correlation spectroscopy (FCS): FCS measurements are carried out using confocal optics to provide the highly focused excitation light and background rejection required for single molecule detection. Fluorescence intensity distribution analysis (FIDA): It yields information on changes in spectral shift, and can also be used to monitor binding events when the binding interaction influences these properties.

14. CELLULAR ASSAY

15. DATA ANALYSIS AND MANAGEMENT Owing to the large volume of data generated in HTS efficient data management is essential. Software packages for HTS (e.g. Activity base , spotfire ) are available to carry out the principle Tasks like A) storage of raw data B) quality control C) transformation of data into information D) documentation E) reporting In HTS each biochemical experiment in a single well is analyzed b y an automated device, typically a plate reader or other kind of detectors.

16. The output of these instruments comes in different formats depending on the type of reader. Sometimes multiple readings are necessary, and the instrument itself may perform some initial calculation. These heterogeneous types of raw data are automatically transferred into the data management software. In the next step raw data are translated in contextual information by calculating results. Data on percentage inhibition or percentage of control are normalized with values obtained from the high and low controls present in each plate. Values obtained depends on the method used (e.g. fitting algorithms used for dose-response curve) and have to be standardized for screens with a company. All the plates that fail against one or more quality criteria are discarded. A final step in the process requires the experimenter to monitor visually the data that have been flagged, as a final check on quality. This is to ensure the system has performed correctly. In addition to registering the test data, all relevant information about the assay has to be logged, e.g. the supplier of reagents, storage conditions, a detailed protocol, plate layout, and algorithms for the calculation of results. Each assay run is registered and its performance documented. HTS will initially deliver hits in targeted assays. Retrieval of these data has to be simple.

17. HIGH THROUGHPUT LIBRARY SCREENING Libraries usually consist of micro titer plates coating frozen or dried samples of compound perhaps only microgram per well One can reduce the screening effort by pooling groups of structure & running assays on mixtures of compounds The factors that limit the pooling are Ionization Reactivity Solubility To be effective a given compound must dissolve completely in the assay medium. It is common to add a small amount (1%) of dimethyl sulfoxide to the assay to assist solvation.

18. MEASUREMENT OF ACTIVITY IN HTS The method must be accurate, reproducible & have high signal to noise ratio. There are 2 methods NON RADIOMETRIC RADIOMETRIC NON RADIOMETRIC : this method include absorbance, fluorescence & luminescence spectroscopy. The assay is usually run at or below the Km value of substrate with only about 5% of substrate consumed during the assay & multiple enzyme turnovers occur during the assay.

19. RADIOMETRIC METHOD : this includes filtration, scintillation proximity assay (SPA). In filtration assay a radioactive substrate bound to a capsule group is cleaved by its enzyme removing the radioactivity from the capture group. The mixture is filtered through special filter paper that the capture group sticks to the filter paper. A scintillation fluid is added & the radioactivity of filter is measured . The degree to which the radioactivity is retained measures the strength of the inhibition. Scintillation proximity assay method: SPA is a newer , simpler method . Started with the same radioactive substrate which may not necessary need a capture group. The enzyme & potential drugs are added , causing the cleavage of substrate to some degree . Now instead of filtering a special resin bead coated with a scintillant ( a compound that fluorescence in the presence of radioactive substrate in the mixture). The lysed and unlysed substrate binds to the beads & if the radioactive part of substance is still attached the bead will fluorescence.

20. SCREENING TECHNOLOGY REQUIREMENTS OF SCREENS: Screens designed to test natural products must be sensitive, selective & able to test large number of samples. Use of appropriate technology to achieve a lower detection limit of 10-200nm is important because the concentration of metabolites in each library sample is unknown, so it is important to detect potent compounds present in low concentrations. E.G. Concentration of metabolites is 1-10 microgram/ml in crude extract have avg. Molecular weight of 500 da & diluted to 20-200 fold in assay the required detection limit of screen is in 10-200nm range. Screens should be specific also for molecular of cellular disease target of choice the data generated from all screens in which samples have been tested should be compared so that selective HTS can be identified at an early stage.

21. This combination of specific screens, data comparison & discriminatory assays makes possible the selection of the best hits . The screening system must be compatible with physiochemical characteristics. Hence, natural products screens are operational in the presence of solvents, are buffered against extremes of ph. & ionic strength & are not affected by color. 2. HTS & SECLECTION FOR PLANT MATERIALS The actual impact of HTS and the suitable selection for plant materials always prevails predominantly In the event when a ‘target’ belongs exclusively to a ‘specific class’ that is rather difficult to find Small molecule hits, such as: ( A ) protein-protein interactions, ( B ) occurrence of a strong precedent, and ( C ) rationale for natural product-derived activities. Importantly, the complete compliance of the above three aspects legitimately command the Desired natural product input.

22. Examples: the following typical examples would be further useful in the adequate clarifications Of the above statement of facts: ( A ) antimicrobial activity i.e. , The track-record of various drug discoveries from a wide spectrum of microbial sources, and ( B ) analgesic medicaments i.e. , The same rationale shall hold good for the adequate track record of plant species in the production of analgesic medicaments. Salient features: the various salient features with regard to HTS and selection for plant materials are as stated under: (1) an easy and convenient access to diversified and huge collections of natural plant materials. Examples: ( A ) samples that are collected skilfully in order to add varying diversity to the important Collection,

23. ( B ) collections may include such samples that are specifically selected based upon various logical reasons viz. , Microbial producer of a certain chemical entity, or a plant prominently employed ethnomedically for a certain prescribed parameter. (2) a diversity-based point of view certainly requires adequate gaining possession of pre-selected taxonomic groups. Thus, one may make use of a variety of time-tested techniques to critically analyse the natural taxonomic spread of a plant collection which may subsequently be extended to minimise the existing gaps so that the ultimate collection distinctly reflects the ‘available diversity’ more exhaustively. (3) ‘chemical targeting’ and ‘biological targeting’: recently, a much more critically focused approach exclusively based upon the ‘prior available knowledge pertaining to some selected samples’ amply suggests that they invariably comprise of a good number of: ( A ) highly specific chemical classes of interest, and ( B ) essentially possess desirable biological characteristic features.

24. Interestingly, the aforesaid ‘approach’ may be justifiably considered under two categories, Namely: ( i ) Chemical Targeting: It accomplishes its cardinal objectives in two different manners, namely: makes use of natural plant materials as the prime sources of particular chemical compounds of great interest to a specific disease regimen, and provides genuine and authentic sources of chemical class of compounds predicted to possess appropriate ‘pharmacophore moieties’. ( ii ) Biological Targeting: It may be regarded as to per sue a disease driven process. In actual practice, one may even select plant samples that may be utilized for the ‘biological evaluation 'thereby providing some sort of relevant information associated with them which in turn could throw ample light with respect to their precise relevance for evaluation of given therapeutic target.

25. Examples: ( a ) the ethnobotanical reports of traditional medicinal applications of plant materials, and ( b ) commercially available orthodox medicinal duly discovered by definite leads given by indigenous knowledge.

26. Strategies Adopted for Identification Process of Plants for Targeted Sets S.No Research Group Adopted Methodology Comments 1 Ethnobotanical Network Worked intimately with indigenous colleagues and traditional doctors in different countries. Low output of actual plant samples for evaluation in laboratory. High output of valuable information(s) of their usage. 2 Pharmaceutical Companies Make use of information(s) reported in Books, Journals etc. Chinese Traditional Medicines; Indian Traditional Medicines 3 Natural Products Alert [NAPRALERT] , Database The system is maintained at the University of Illinois at Chicago (USA) Contains huge number of references related to Ethnobotanical reports Reports of biological activity in scientific literature, and Phytochemical data. 4 Chemical Information Databases ] Dictionary of Natural Products [Chapman and Hall, New York Database contains information on more than 100,000 natural plant products, including the plant species from where the chemical compound actually originates. 5 Literature Survey To generate semi-purified plant extracts or chemical group of specific interest, or extracts that are enriched in the chemical entities Plants having an ethnomedical application the extracts may be prepared using recommended traditional medicine

27. APPLICATIONS In the recent past, the discovery of a plethora of extremely potent and elegantly novel euphane triterpenes amply proves and demonstrates the actual enormous ability and potential of several plant extracts to produce highly beneficial ‘chemical leads’ in a defined HTS-programme. The above factual observations may be duly substantiated with the help of the following important investigative experimental results, namely: ( a ) Inhibitors of Human Thrombin: In the usual course of a ‘random’ screening exercise to look for certain ‘novel inhibitors of human thrombin’ that essentially help in the critical blockade of the actual formation of blood clots; and, therefore, may be duly exploited in the treatment, control, and prevention of the deep-vein thrombosis. For this meticulous task, a sizable (approx. 1,50,000) samples adequately derived from both natural sources viz. , plant extracts, microbial extracts, fungal extracts, and purely synthetic chemical compounds were subjected to vigorous investigative evaluations.

28. The interesting outcome of this big-job revealed that the methanolic extracts of Lantana camara leaves , belonging to the natural order Verbenaceae, showed remarkable potent activity. ‘lantana poisoning’ is associated with the following vital biological effects, such as: Enhancement in blood-coagulation time and prothrombin time, Reduction in blood-sedimentation rate, Total plasma- protein content, and Total fibrinogen content. The aforesaid observation ascertains the corresponding thrombin-inhibitory translactone having the euphane triterpenes. ( b ) Biological Activity of the euphane triterpenes The investigative study amply revealed their actual ‘mechanism of action as specific inhibitors for blood clothing through the strategic acylation of the available active site(s)

29. A high-throughput mapping and sequencing of Gangliosides in human foetal brain was performed by a novel mass Spectrometry (MS)-based approach. Three GG mixtures extracted And purified from different regions of the same foetal brain in the 36 th gestational week: frontal neocortex (NEO36), white matter Of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening And multi-stage fragmentation by fully automated chip-based Nano electrospray ionization (nanoesi) high capacity ion trap (HCT) MS.Using this method, in only a few minutes of signal acquisitions, over 100 GG species were detected and identified in the three mixtures. Penicillin G acylase (pga) is one of the most important enzymes for the production of semisynthetic β-lactam antibiotics and their key intermediates. Purification of penicillin G acylase from fermentation broth with the aid of high-throughput screening (HTS) process was recently studied to speed up the process. Micro titer -plate was used for screening method to find appropriate purification conditions for the target protein. The screening method is based on a 96-well plate format.

30. HIGH TECH PRODUCTS FROM NATURAL RESOURCES: 1. TAXANES: baccatin & 10- deacetylbaccatin have been effectively transformed into taxol & obtained from twigs and leaves of Taxus Baccata 2. GENISTEIN: Genistein is the phytoestrogen normally found in soy products. It represents the aglucon of Genistein and of sophoricoside. It serves as a specific protein kinase inhibitor. Genistein causes the inhibition of angiogenesis, steroid hormone receptors, inhibition of tyrosine kinase, inhibition of radical O2-species formation, and above all interaction with topoisomerase.

31. CONCLUSION The HTS field continues to dynamic and extremely competitive one, where a newer technique or method is being reported at a very frequent basis. The need to increase the throughput of drug-discovery screening operations while reducing development and operating costs is continuing to drive the development of homogeneous, fluorescence-based assays in miniaturized formats. The use of 384-well and higher density plates and commercially available plate-handling robotics has made HTS a reality, and has allowed some screening groups to achieve ultra-high throughput rates in excess of 100,000 samples per day. As the density of plate increases the volume of sample required for the assay is decreased drastically, as a result the assay of expensive drugs can be carried out at lower cost, which compensates the initial setup cost. The combination of Nano litre scale liquid-handling, integrated devices for compound dilution and assay functionality, and state-of-the-art fluorescence detection techniques has the potential to revolutionize the drug discovery screening process.