1. High throughput screening (HTS) is a process for screening large numbers of biological compounds against selected targets using automated equipment. It aims to accelerate the drug discovery process. 2. The key steps in HTS include target identification, reagent preparation, assay development, screening compound libraries, data analysis and management. HTS assays can be biochemical, cellular, or involve measuring second messengers. 3. HTS has various applications in natural product drug discovery, including identifying inhibitors of human thrombin from plant extracts. Euphane triterpenes isolated from Lantana camara leaves showed potent thrombin inhibitory activity.

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
ORGANISM
COLLECTION
{Targeted
fermentation,
stringent extraction}
SAMPLE/
COMPOUND
{Targeted
identification ,
Development}
HIGH THROUGHPUT
{Screen technology,
robotics , data}
CHEMICAL
INVESTIGATION
{Biological profiling ,
development & scale-
up , elucidation}
CHEMICAL
{Rapid selection of
quality}
PRIMARY HITS
{Multi-parametric
criteria , advanced
informatic}
QUALITY DRUG
LEADS
{Chemical
modification,
biological evaluation,
clinical}
LEAD
OPTIMIZATION/
DRUG
DEVELOPMENT

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:
1. Target identification
2. Reagent preparation
3. Assay development
4. Data analysis & management
5. 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.
1. 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.
2. 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
BIOCHEMICAL
ASSAY
HOMOGENEOUS
ASSAY
NON HOMOGENEOUS
CELLULAR OR
CELL BASED
ASSAY
CELL PROLIFERATION
ASSAY
RECEPTOR GENE
ASSAY
SECOND
MESSENGER ASSAY

12. BIOCHEMICAL ASSAY
HOMOGENEOUS
ASSAY
REAGENTS ARE ADDED ,
INCUBATED & EFFECT OF
COMPOUNDS IS MEASURED
WITHOUT THE NEED TO INCLUDE
A SEPRATION STAGE
NON HOMOGENEOUS
ASSAY
IT INVOLVS A
SEPRATIONSTAGE

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
CELL
PROLIFERATION
ASSAY
IT MONITORS THE
OVERALL GROWTH
RESPONSE OF CELLS TO
EXTERNAL STIMULI
RECEPTOR GENE
ASSAY
MONITORS CELLULAR
RESPONSES AT
TRANCRIPTION OR
TRANSLATIONAL LEVEL
SECOND
MESSENGER
MONITORS SIGMA
TRANSDUCTION
FOLLOWING ACTIVATION OF
CELL SURFACE RECEPTORS

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 by 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
1. Ionization
2. Reactivity
3. 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
1. NON RADIOMETRIC
2. 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).
1. 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.
2. 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
1. 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 36th
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.