Combinatorial chemistry is a collection of techniques which allow for the synthesis of multiple compounds at the same time. Combinatorial chemistry is one of the important new methodologies developed by researchers in the pharmaceutical industry to reduce the time and costs associated with producing effective and competitive new drugs, By accelerating the process of chemical synthesis, this method is having a profound effect on all branches of chemistry, but especially on drug discovery.

1. Combinatorial chemistry and High-
Throughput Screening
Presented by :
Saikiran kulkarni
KLE college of pharmacy
Depatment of pharmacology and toxicology

2. Introduction and definitions
• Combinatorial chemistry is a collection of techniques which allow for the synthesis of
multiple compounds at the same time.
• Combinatorial chemistry is one of the important new methodologies developed by researchers
in the pharmaceutical industry to reduce the time and costs associated with producing
effective and competitive new drugs, By accelerating the process of chemical synthesis, this
method is having a profound effect on all branches of chemistry, but especially on drug
discovery.
• Through the rapidly evolving technology of combinatorial chemistry, it is now possible to
produce compound libraries to screen for novel bioactivities.
• In combinatorial approach, one can cover many combinations An x Bn in one reaction Instead
of doing multiple A x B type reactions

3. • Combinatorial chemistry may be defined as the systematic and repetitive, covalent connection
of a set of different “building blocks” of varying structures to each other to yield a large array
of diverse molecular entities
• Combinatorial chemistry encompasses many strategies and processes for the rapid synthesis
of large, organized collections of compounds called libraries. The collection is then tested for
the biological activity. Finally the active compound is identified and made in quantity as a
single compound.
• Thus the combinatorial chemistry approach has three phases:
1. Making a library. 2. Finding the active compound. 3.Screening mixtures for biological activity
Hence, In combinatorial chemistry, large numbers
of compounds are made at the same timein small
amounts, forming libraries which can be assayed
for desired properties all at once. Finally the
active compound is identified and made in
quantity as a single compound.

5. 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.
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

6. Disadvantages
• Though combinatorial chemistry would solve all the problems associated with drug
discovery, one still needs to synthesize the right compound.
• There is a limit to the chemistry you can do when using solid phase synthesis. The
resin you use is often affected by the reaction types available and care must be
taken so that the attachment of the reagent to the substrate and bead are unaffected.
Each reaction step has to be carefully planned, and often a reaction isn't available
because the chemistry affects the resin.
• While a large number of compounds are created, the libraries created are often not
focused enough to generate a sufficient number of hits during an assay for
biological activity.
• Efficiency is highly affected by compound's size, solubility and function group.
• Compounds produced tend to be Achiral of Racemic

8. Principle of combinatorial chemistry
• The basic principle of combinatorial chemistry is to prepare a large
number of different compounds at the same time Instead of synthesizing
compounds in a conventional one at a time manner and then to identify
the most promising compound for further development by high
throughput screening.
• The characteristic of combinatorial synthesis is that different compounds
are generated simultaneously under identical reaction conditions (ie. using
the same reaction conditions and the same reaction vessels.) in a
systematic manner, so that ideally the products of all possible
combinations of a given set of starting materials (termed building blocks)
will be obtained at once. The collection of these finally synthesized
compounds is referred to as a combinatorial library. The library is then
screened for useful properties and the activecompounds are identified

9. The combinatorial libraries can be
structurally related by a central core
structure, termed scaffold(ie. all
compounds of library have a common
core structure),or by a common
backbone.

10. Method involved in combinatorial synthesis
There are two approach by which the combinatorial libraries can be generated:
1)Biological library approach
a)Filamentous phage approach
b)Plasmid approach
c)Polysome approach
2)Spatially addressable parallel solid phase library approach
a)Multi-pin methodology
b)Tea bag methodology
c)SPOTS membrane method
d)Light directed peptide synthesis on resin support
Combinatorial chemistry can be applied to :
-Solid phase combinatorial chemistry
-Solution phase combinatorial chemistry

11. 1)Biological approach to generate molecular diversity :
• The use of biological system for the generation of peptide diversity mimics
the evolution creation of protein diversity. Artifical evolution can be greatly
enhanced by the introduction of diversity in to the system at a much higher
rate than that occurs naturally. The source of the diversity in the
combinatorial chemical synthesis is the structure of oligonucleotides.
Oligonucleotide synthesis is a well characterized chemistry that allows
tight control of the composition of mixture created. The degenerated
sequence produced are then cloned and expressed as peptides.

12. 2)Spatially addressable parallel library approach
• The desire to develop and explore SAR around peptide lead compound has placed
tremendous demands on the productivity of peptide chemistry.
a)Multiple pin methodology
• In this method,the synthesis, of peptides takes place on polyethylene pins (4×40 mm)
functionalized with acrylic acid arranged in 96 well formats.The well contain
activated amino acid monomers.Peptide synthesis is carried out at the end of a spacer
(e.g.NB-Fmoc-β-alanyl-1,6-diaminohexane).Screening is done by means of enzyme
linked immunosorbent assay (ELSIA) to determine the binding capability of
covalently bound peptide to antibodies.

13. Tea-bag synthesis
• Introduced by Richard
Houghten (1958). Rapid
multiple peptide
synthesis,Polyethylene bag
with fine holes (similar to tea
bag) filled with resins. Each
bag is put in the different
reaction vessels to carry out
amino acid coupling reaction.
• After that, all bags are
collected and processed
together for protecting group
removing and washing resins
(to reduce time and efforts)
• Bag act as filter and prevent
resin mixing between
reactions and by labeling
each bag, the synthesised
peptide structure can be
identified

14. SPOTS membrane method
• This method introduced by Frank (1992) and his group also for preparing peptide arrays.
The synthesis is carried out on cellulose paper membranes instead of polyethylene pins
as the solid support for peptide synthesis. Small droplets of solutions of protected amino
acids dissolved in low volatility solvents & coupling reagents are pipetted on to
predefined positions of the membrane. The spots thus formed can be considered as
reactors when the conversion reactions of the solid phase synthesis take place
• An array of as many as 2000 peptides can be made on a8 x 12 cm paper sheet. The
peptides can be screened on the paper after removing the protecting groups.

15. Light directed spatially addressable parallel chemical synthesis
• The basic principle of the method is that each set of building blocks contains a
photolabile protecting group (NVOC) and only the building blocks which have
been exposed to light can be coupled with another building block. Thus, pattern of
masks and the sequence of protecting groups define the final structure of the
compounds synthesized.
• The light directed method makes it possible to prepare an array of peptides or other
kinds of molecular on the surface of a glass slide. The surface of the glass is
functionalized with amino alkyl groups protected by the photolabile 6–nitro Vera
tryloxy carbonyl (NVOC) groups. The amino acid used in the synthesis arealso
protected by NVOC. group.

16. SOLID PHASE COMBINATORIAL CHEMISTRY :
• In solid phase combinatorial chemistry, the starting compound is attached to an insoluble resin bead,
reagents are added to the solution in excess, and the resulting products can be isolated by simple filtration,
which traps the beads while the excess reagent is washed away.
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
• Individual beads can be separated to isolate individual products
• Polymeric support can be regenerated and re-used after cleaving the product
• Automation is possible

17. Requirements :
• A resin bead or a functionalised surface to act as a solid support
• An anchor or linker
• A bond linking the substrate to the linker. The bond must be stable to the
reaction conditions used in the synthesis
• Protecting groups for functional groups not involved in the synthesis

19. Solid support used in Solid phase synthesis :
• Most solid state combinatorial chemistry is conducted by using polymer beads
ranging from 10 to 750 μm in diameter. The solid support must have the following
characteristics for an efficient solid-phase synthesis:
1) Physical stability and of the right dimensions to allow for liquid handling and
filtration
2) Chemical inertness to all reagents involved in the synthesis
3) An ability to swell while under reaction conditions to allow permeation of solvents
and reagents to the reactive sites within the resin
4) Derivatization with functional groups to allow for the covalent attachment of an
appropriate linker or first monomeric unit

20. TYPES OF SOLID PHASE USED:
• Polystyrene resins: In this, Polystyrene is cross linked with divinyl benzene (about
1% cross linking). Polystyrene resin is suitable for non-polar solvents.
• Tenta Gel resins: It consists of about 80% polyethylene glycol (PEG) grafted to
cross-linked polystyrene.It Combines the benefit of the soluble polyethylene glycol
support with the insolubility & handling characteristics of the polystyrene bead.
• Poly acrylamide resins: Like super blue these resin swell better in polar solvent,
since the contain amide bonds, more closely resemble biological materials.
• Glass and ceramic beads: These type of solid supports are used when high
temperature and high pressure reaction are carried out.

21. Linkers and anchors used in solid phase synthesis:
• The initial building block of the compound to be
prepared by solid phase synthesis is covalently
attached to the solid support via the linker.
• The linker is a bifunctional molecule. It has one
functional group for irreversible attachment to the
core resin and a second functional group for forming
a reversible covalent bond with the initial building
block of the product.
• The anchor can also be considered as a protecting
group of one of the functional groups of the final
product and, as such, it determines the reaction
conditions by which the product can be cleaved from
the support.

22. PROTECTING GROUPS USED IN SOLID PHASE SYNTHESIS :
• A protecting group is reversibly attached to the functional group to convert it to a less reactive
form. When the protection is no longer needed, the protecting group is cleaved and the
original functionality is restored. A large number of protecting groups were developed for use
in peptide synthesis since the amino acids are multifunctional compounds.
(If a chemist wants to carry out a reaction on only one functional group of a multifunctional group compound,
the reactivity of the rest of the functional groups needs to be suppressed)
The benzyl carbonyl (Z) group The t-butoxy carbonyl (Boc) group
9-fluorenyl methoxy
carbonyl (Fmoc) group
Protection of carboxyl groups by
(A) benzyl esters and (B) t-butyl
esters

24. SOLUTION PHASE COMBINATORIAL CHEMISTRY:
• The solution phase synthesis involves conducting chemical reaction simultaneously,
preferably in well-ordered sets (arrays) of reaction vessels in solution. Most ordinary
synthetic chemistry takes place in solution phase. The use of solution phase techniques has
been explored as an alternative to solid-phase chemistry approaches for the preparation of
arrays of compounds in the drug discovery process. Solution phase work is free from some of
the constraints of solid-phase approaches but has disadvantages with respect to purification.
• In solution phase synthesis we use soluble polymer as support for the product. PEG is a
common vehicle which is used in solution phase synthesis it can be liquid or solid at room
temperature and show varying degrees of solubility in aqueous and organic solvent. By
converting one OH group of PEG to methyl ether (MeO-PEG-OH) it is possible to attached a
carboxylic acid to the free OH and use in solution phase combinatorial synthesis
• Another common support which is used in solution phase synthesis is liquid Teflon consisting
mainly of long chain of (-CF2 -) groups attached to a silicon atom. When these phases are
used as a soluble support for synthesis the resulting product can be easily separated from any
organic solvent.
• The main disadvantage of this method is when number of reagents are taken together in a
solution, it can result in several side reactions and may lead to polymerization giving a tarry
mass. Therefore, to avoid this, the new approach is developed in which all chemical structure
combinations are prepared separately, in parallel on a giving building block using an
automated robotic apparatus.

25. Split and Mix Synthesis Method:
• In this method, ingredients are assembled on
the surface of the beads or micro particles. In
each step, beads from last steps are
partitioned into new building block and
several groups are added. This leads to the
formation of new groups, the different groups
of beads are recombined and separated once
again. Process is continuous with next
building block is added until the desired
library has been assembled

26. High-Throughput Screening
• The mechanism-based approach which corresponds to the target-based approach
screens for compounds with a specific mode of action. The highly effective nature of
high-throughput screening (HTS) for identification of highly target specific compounds
is attributed to its precise focus on single mechanism.
• The vast number of compounds produced by combinatorial chemistry and the
possibility of testing many compounds, including natural products, in a short period of
time by HTS attracted attention of many workers
• Various detection techniques like fluorescence resonance energy transfer (FRET),
Homogeneous time resolved fluorescence (HTRF), etc are available, and the screening
of more than 100,000 samples per day is possible. With the introduction of robotics,
automation and miniaturization techniques, it became feasible to screen 50,000
compounds a day with complex work-stations
• High-throughput screening methods are also used to characterize metabolic and
pharmacokinetic data about new drugs. With the use of Cassette dosing techniques even
the pharmacokinetic data can be assessed for large number of drug candidates.

27. • The main purpose or goal of this technique is to hasten the drug discovery process by
screening the large compound libraries with a speed which may exceed a few thousand
compounds per day or per week. For any assay or screening by HTS to be successful several
steps like target identification, reagent preparation, compound management, assay
development and high-throughput library screening should be carried out with utmost care
and precision.
• High-Throughput Screening is a very vast area of study and development with many scopes
including topics like enzyme testing, whole organ testing and even whole animal testing via
cassette dosing. Cassette dosing is a procedure for HTS enabling to rapidly assess the
pharmacokinetics of large number drug candidates.

28. • HTS is a novel method for drug discovery but it is not the only method. HTS
not only helps in drug discovery but also in development of present drug
moieties to optimize their activity
• In past years many advances in science and technology and economic pressures
have kept every researcher to develop speedy and precise drug development
and screening technologies to combat the ever increasing diseases and many
pathogens acquiring resistance to present available drugs.
• It is anticipated that with the introduction of human genomes as potential
candidates the compound library will be as large as 100 million candidates that
would require about 1012 assays to establish their Structure-Activity
Relationship (SAR).
• Initially the assays were carried out in 96-well plates but with advancement
now there are also 1586-well plates available. Typical HTS programs have
potentials to screening up to10000 compounds per day, while some laboratories
with Ultra High-Throughput Screening (UHTS) can perform 100,000 assays
per day

29. ASSAY DESIGN :
• If a positive result or “HIT” is discovered in primary screen a more accurate and precise
secondary screening is performed and accordingly quantification and IC50 calculation are
done. The assay procedures are no different from those known to biological and
biochemical scientists like ELISA, reporter-gene assays binding assays etc.
• Assays are mainly of two types either heterogeneous or homogeneous. Heterogeneous
assays are bit complex requiring additional steps like filtration, centrifugation etc. beyond
the usual steps like fluid addition, incubation and reading. Homogeneous assays are
simpler consisting of the latter three usual steps, this may also be called true homogeneous
assay.
BIOCHEMICAL ASSAYS:
• Homogeneous biochemical assays in miniaturized formats are most frequently carried out
using scintillation proximity assay (SPA) or fluorescence detection techniques because of
the requirement for increased sensitivity as assay volume shrinks. The choice of detection
technology employed is dependent on the particular class of assay target being
investigated. Because of the high binding and low receptor densities required, binding
assay for cell surface receptors are usually carried out using SPA techniques.

30. Techniques of biochemical assays :
• Fluorescence resonance energy transfer (FRET): It is the non-radioactive
transfer of energy between appropriate energy donor and acceptor molecules.
• Homogeneous time resolved fluorescence (HTRF): It is a hybrid technique
that takes advantage of the long fluorescence lifetimes of europium crytates and
the large apparent Stokes shift (the difference between the peak excitation and
peak emission wavelengths of a fluorophores) obtained by exploiting energy
transfer between the europium donor and suitable acceptors.
• 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 fluorophore quantum yield or spectral shift, and can also be used to
monitor binding events when the binding interaction influences these properties

31. Cell-based assays :
• Cell-based assays refer to any of a number of different experiments based on the
use of live cells
• This is a general definition and can include a variety of assays that measure cell
proliferation, toxicity, motility, production of a measurable product and
morphology
• Cell-based assays offer a more accurate representation of the real-life model
since live cells are used
FOUR KEY ELEMENTS OF CELL BASED ASSAY:
•A cellular component e.g. a cell line or a primary cell population
• A target (substrate) molecule that records the cellular response
•An instrument to conduct and monitor the assay & an informatics component to manage and
analyse data from the assay
•Cell-based reporter assays are used where humanreceptors are transfected into null cell lines
eitheralone, or as part of receptor systems constructed toshow alterations in light production
(luciferinluciferase)or light transmission (melanophore),that can be measured independently of
radioactivity within minutes

32. Advantages over biochemical assay
• Assays do not require purification of the target protein
• Can immediately select against compounds / potential drugs that are generally
cytotoxic, or that cannot permeate cellular membranes to reach intracellular
sites
• Hit/lead compounds identified by cell based assays have passed important
validation steps, saving time and costs in drug development
• Cell-based assays visualize all possible drug-target interactions e.g. activators,
target interactions

33. Cell-based assays :
Second messenger assays: It monitors signal transduction
from activated cell-surface receptors. Second messenger
assays typically measure fast, transient fluorescent signals
that occur in matter of seconds or milliseconds. 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 of second messenger assays for receptor
stimulation and ion-channel activation.
Reporter gene assays: It monitors cellular responses at
transcription/translation level. It indicates the presence or
absence of a gene product that in turn reflects changes in a
signal transduction pathway. The quantification of the
reporter is usually carried out by biochemical methods viz
by measuring the enzymatic activity. Plasmids are typical
reporter genes employed.

34. • Yeast complementation assay: S. cerevisiae is the typical model
organism used for the two-hybrid technique's inception. It has
several characteristics that make it a sturdy organism to host the
interaction, including the ability to form tertiary protein structures,
neutral internal pH, enhanced ability to form disulfide bonds and
reduced-state glutathione among other cytosolic buffer factors, to
maintain a favourable internal environment. Yeast systems are
tolerant of diverse culture conditions and harsh chemicals which
cannot be applied to mammalian tissue cultures. Proteins from as
small as eight to as large as 750 amino acids have been studied using
yeast cultures.
• Cell proliferation assays: It monitors the overall growth/no growth
responses of the cell to external stimuli. These are quick and easy to
be employed for automation

35. Refrences
• Martis EA, Radhakrishnan R, Badve RR. High-
throughput screening: the hits and leads of drug
discovery-an overview. Journal of Applied
Pharmaceutical Science. 2011;1(1):2-10.
• Narang H, Shastri S. Combinatorial Chemistry-
Modern Synthesis Approach. PharmaTutor. 2017
May 1;5(5):37-63.