Combinatorial chemistry and high-throughput screening techniques allow for the rapid synthesis and testing of large libraries of compounds. Combinatorial chemistry uses solid and solution phase methods to efficiently produce thousands of compounds, while high-throughput screening employs automated instrumentation like microtiter plates to quickly assess large numbers of compounds through functional or non-functional assays. These approaches provide advantages for drug discovery by facilitating the identification of hit compounds for further optimization into drug leads.

1. METHODS AND ADVANTAGES IN COMBINATORIAL
CHEMISTRY AND HIGH THROUGHPUT SCREENING
PRESENTED BY
ANJI REDDY
Y17MPHPY454
DEPARMENT OF
PHARMACOLOGY

2. CONTENTS
• COMBINATORIAL CHEMISTRY
• DIFFERENCE BETWEEN TRADITIONAL
SYNTHESIS & COMBINATORIAL SYNTHESIS
• DRUG DISCOVERY
• TECHNIQUES USED IN COMBINATORIAL
CHEMISTRY
• ADVANTAGES

3. Combinatorial chemistry
• Combinatorial chemistry comprises chemical synthetic methods that make
it possible to prepare a large number (tens to thousands or even millions) of
compounds in a single process. These compound libraries can be made as
mixtures, sets of individual compounds or chemical structures generated by
computer software. Combinatorial chemistry can be used for the synthesis
of small molecules and for peptides.
• Strategies that allow identification of useful components of the libraries are
also part of combinatorial chemistry. The methods used in combinatorial
chemistry are applied outside chemistry, too

4. Difference Between Traditional
Synthesis & Combinatorial Synthesis

6. DRUG DISCOVERY

7. TECHNIQUES USED IN COMBINATORIAL
CHEMISTRY
• solid phase Technique
 Solid Support Method
 Parallel Synthesis
 Manual method
 Automated
 Mixed combinatorial Synthesis
 Mixed & split Combinatorial Synthesis
• Solution phase Technique

8. SOLID PHASE TECHNIQUES
• Reactants are bound to a polymeric surface and modified
whilst still attached. Final product is released at the end of the
synthesis
Advantages
• Specific reactants can be bound to specific beads
• Beads can be mixed and reacted in the same reaction vessel
• Products formed are distinctive for each bead and physically
distinct
• Excess reagents can be used to drive reactions to completion
• Excess reagents and by products are easily removed
• Reaction intermediates are attached to bead and do not need to
be isolated and purified

9. Examples of Solid Supports
• Partially cross-linked polystyrene beads hydrophobic in nature
• causes problems in peptide synthesis due to peptide folding
• Sheppard’s polyamide resin - more polar
• Tentagel resin - similar environment to ether or THF
• Beads, pins and functionalised glass surfaces

10. Solid phase synthesis: protecting groups
• A few protecting groups used in solid phase synthesis.
 For amines.
• Boc ( t-butoxycarbonyl )
• Fmoc (9-fluorenylmetoxy carbonyl)
• Tmsec (2 [ trimethylsilyl ] ethoxycarbonyl)
 For carboxylic acids.
• Tert Bu ester(t-butyl ester)
• Fm ester(9-fluronyl methyl ester)
• Tmse ester(2 [trimethylsilyl] ethyl)

11. Parallel Synthesis
• To use a standard synthetic route to produce a range of
analogues, with a different analogue in each reaction vessel,
tube or well
• The identity of each structure is known
• Useful for producing a range of analogues for SAR or drug
optimisation

12. Houghton’s Tea Bag Procedure
• Each tea bag contains beads and is labelled
• Separate reactions are carried out on each tea bag
• Combine tea bags for common reactions or work up
procedures
• A single product is synthesised within each tea bag
• Different products are formed in different tea bags
• Economy of effort - e.g. combining tea bags for workups
• Cheap and possible for any lab
• Manual procedure and is not suitable for producing large

13. Automated parallel synthesis
• Automated synthesisers are available with 42, 96 or 144
reaction vessels or wells
• Use beads or pins for solid phase support
• Reactions and work ups are carried out automatically
• Same synthetic route used for each vessel, but different
reagents
• Different product obtained per vessel

14. Mixed Combinatorial Synthesis
• To use a standard synthetic route to produce a large variety of
different analogues where each reaction vessel or tube contains
a mixture of products
• The identities of the structures in each vessel are not known
with certainty
• Useful for finding a lead compound
• Capable of synthesizing large numbers of compounds quickly
• Each mixture is tested for activity as the mixture
• Inactive mixtures are stored in combinatorial libraries
• Active mixtures are studied further to identify active
component

15. Mix and Split Method
• Synthesis of all possible dipeptides using 5 amino acids
• Standard methods would involve 25 separate syntheses
• Combinatorial procedure involves five separate syntheses
using a mix and split strategy

17. Solution phase Technique
• It is the modified reaction to accommodate a solid support .
• Solution phase combinatorial chemistry often lead to a
formation of Mixture of product .
• May helpful for development of Amazing Mixture
Problems :
1. difficulty of removing unwanted
material
2. purification at each step is necessary
3. other practical problem

18. ADVANTAGES
 Fast
• Combinatorial approach can give rise to million of compound in
same time as it will take to produce one compound by traditional
method of synthesis .
 Economical
• A negative result of mixture saves the effort of synthesis,
purification & identification of each compound
 Easy
• Isolation purification & identification of active molecule from
combinatorial library is relatively easy.

19.  Drug Discovery
• Mixed Combinatorial synthesis produces chemical pool.
• Probability of finding a molecule in a random screening
• process is proportional to the number of molecules
subjected to the screening process
 Drug Optimization
• Parallel synthesis produces analogues with slight
differences
• which is required for lead optimization

20. HIGH THROUGHPUT
SCREENING

21. CONTENTS
• INTRODUCTION AND SHORT HISTORY
• WHAT IS HTS..?
• INSTRUMENTATION
• TECHNIQUES AND PROCEDURE
• IMPORTANCE AND APPLICATIONS OF HTS
• LIMITATIONS OF HTS

22. INTRODUCTION AND SHORT HISTORY
• In order to learn about high throughput screening, we should
have a general understanding of the drug discovery process.
• Drug discovery is the process by which new medications or
drugs are discovered.
• It involves the various steps from disease identification,
identification and validation of related target(s), development
of a lead that can interact with the target and be effective in
treating the disease, preclinical and clinical trials and then
finally marketing.
• High throughput Screening was invented by Dr. Gyula
Takatsky in 1951; he made the first microtiter plate using
Lucite and creating 6 rows of 12 wells in it.

25. WHAT IS HTS..?
• High throughput screening (HTS) is an experimental process
or tool that employs a group of techniques to quickly conduct a
very vast number of chemical, pharmacological, genetic,
biological tests to identify biomolecular pathways or
pharmacological actions.
• 10,000 – 100,000 compounds can be screened daily.
• Very vital to drug design and drug discovery process, vital to
general scientific and medical research
• Very valuable to early drug discovery

26. • Basically helps to identify a compound that can chemically
modify a target
• This compound is identified as a hit and may be generated to a
lead.
• A Hit is any compound that is confirmed to have binding
activity to the target and appears on High throughput screen. It
gives the desired effect of the HTS experiment and is confirmed
on re-testing.
• The Lead is the compound with therapeutic or pharmacological
activity but suboptimal structure that still requires modification.

27. • This process is commonly referred as lead generation or hit to
lead (H2L).
• The lead is further optimized and thus can then go through
preclinical and clinical trials and if approved gets marketed.
• The general procedure of HTS involves testing a solution of
different compounds in assay plates called microtiter plates
having wells.
• The ligand or protein or embryo of interest is introduced into
these wells containing test solution They are incubated for a
short time period.
• Analysis is done microscopically or by analytical techniques
like spectrometry.
• Compounds showing desired effects are hits.

29. INSTRUMENTATION
 MICROTITER PLATES (ASSAY PLATES)
• Plates/containers made of plastic, having spaced wells – up to
384, 1536 or 3456 wells.
• They would contain solvents (e.g. DMSO + test compounds)
• They would also contain proteins, cells, etc. to be analysed.
• Some might be kept empty or contain pure solvents to serve as
controls.
 DETECTORS
• Diverse spectrometers (fluorescence, mass, NMR, FTIR, etc.),
Chromatography (Gas, Liquid, Ion exchange, etc.) and
Microscopy (Scanning tunnelling microscopy, atomic force
microscopy, confocal microscopy) and Calorimeters.

31. TECHNIQUES AND PROCEDURE
 TYPES OF HTS: Functional and Non-functional.
• Functional: Study exactly how the compound interacts with
targe
• Non-functional: To find out if the compound interacts with
target or not.

32. PROCEDURE

33. Use of robotics
• Robotics and automated systems are and impotent component of
HTS.
• They optimize the process and save manpower.
• Robot arms can be used effectively to transfer microtiter plates to
and fro the sampling, incubation and analysing spots.

34. IMPORTANCE AND APPLICATIONS OF HTS
• Selection of compounds from a vast number synthesized by
combinatorial chemistry and other methods.
• For lead generation for the treatment of a disease.
• It is an efficient tool in studying biomolecular interactions and
pathways.
• It is highly efficient, fast, accurate and dependable in
compound screening
• Useful in DNA sequencing

35. CONT…………………………………….
• Useful in toxicology, to study mechanism of action of various
drugs and toxins.
• Study drug-drug interactions and the effects of drugs on
metabolizing enzymes.
• Useful in cytotoxicity assays
• Useful in genotoxicity assays
• RECENT ADVANCEMENTS
• Use of living organisms in HTS to study drug action and
identify lead molecules e.g. in Zebra fish and Caenorhabditis
elegans.
• Ultra HTS where above 100,000 compounds are screened at a
time; up to 300,000.

36. LIMITATIONS OF HTS
o High cost
o Contamination of samples is possible.
o Analysis of data and selection of relevant data from large moulds of
data requires patience,professionalism, dedication and true expertise.

37. BIBLIOGRAPHY
• An introduction to Medicinal chemistry, rama rao nadendla
• www.slideshare.net/.../combinatorial-chemistry hts and its
applications.
• www.biotech.nature.com ; NATURE BIOTECHNOLOGY ;
Vol 18 ; Supplement 2000.

38. ANJI REDDY