Combinatorial chemistry is a new approach to drug discovery that involves synthesizing and testing large libraries of compounds in parallel rather than one by one. This allows for more rapid and cost-effective discovery of potential drug leads. There are two main challenges in drug discovery that combinatorial chemistry addresses: identifying a lead compound with the desired biological activity, and optimizing the lead compound. Solid phase synthesis and solution phase synthesis are two main combinatorial methods. Case studies demonstrate how combinatorial synthesis approaches have been used to develop inhibitors of influenza endonuclease, kinase inhibitors, and modulators of orexin receptors.

1. Combinatorial
approach for
drug discovery
Shweta Kumari
20172078

2. Combinatorial approach for drug discovery
New approach to drug discovery which results in discovery of interesting potential
pharmaceuticals.
It is the art and science of synthesizing and testing compounds for bioactivity en
masse, instead of one by one, the aim being to discover drugs and materials more
quickly and inexpensively than was formerly possible.
Why Combinatorial approach ?
● Two basic challenge of drug discovery:
○ Identify a lead compound with desired activity
○ Optimize the lead compound to meet the criteria sufficient to proceed with development

3. The long road to a new medicine

4. Structure based drug design knowledge plot

5. Principal characteristics of conventional vs.
combinatorial strategy of drug discovery

6. Developed in the mid 1980’s, with Geysen’s and Houghten’s.
Basic principle : Prepare a large number of similar compounds at the same time
instead of synthesizing compounds in a conventional one at- a-time manner.
Objective : Build a large library of compounds from a starting "scaffold" to interact
with specific biological targets.
Combinatorial library: collection of finally synthesize molecules
Combinatorial chemistry

7. Types of combinatorial library in drug design
Random library
● Multiple library
● Many targets
● Highly diverse
● Mixture
● > 5000 compounds
● Solid phase synthesis
● Non-purified compounds
● On bead screening, if possible
Focused or targeted library
● Template-scaffold library
● One target
● High structural similarity
● Single compounds
● <<5000 compounds
● Synthesis in solution or solid phase
● Pure compounds
● Screening in solution

8. Combinatorial library in drug design
Identification of biological target
Development of an assay
High throughput screening
Hit
Lead structure
Lead optimization
Development
Random libraries
Combinatorial chemistry
Focused libraries

9. Combinatorial Methods
Combinatorialsynthesis
Solid phase
synthesis
Solution phase
synthesis
Tea-bag synthesis
Split-mix technique
Multi-pin synthesis

10. Solid phase synthesis
Source: © The Rockefeller University
● Combinatorial synthesis of peptide on
resin bead by Robert Bruce Merrifield,
American biochemist (1921–2006).
● Nobel Prize in Chemistry, 1984.
● Linear synthesis
● Allow excess of reagents and reactants
to force reaction to completion
● Removal of reagents, reactants and
byproducts by filtration.

11. Number of compounds (peptides) generated by combinatorial
approach
First reports dealt with the
simultaneous production
of collection of chemically
synthesized peptides,
produced by solid phase
methods on solid supports.

12. Parallel synthesis
Compounds are synthesised in separate
vessels but at the same time parallel.
The array of reactions are taken in grid well in
plastic plate (in bead method) or pins (grids of
plastic rods) called crowns.
The building blocks are attached to these
beads or crowns.
The structure of product is identified from the
grid code.

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. Some examples for the use of the tea-bag method:
● Characterization of the influenza haemagglutinin protein (HA1) and discovering
the amino acid position that is critical important to the binding interaction
(Houghten et al. ,1986)
● Production of a small combinatorial library of urea analogues (Burgess et al.,
1997)
● Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)
protected amino acids applied for antigenic mapping of viral proteins.
● Studies on the structural requirement for ligand binding to the neuropeptide Y
(NPY) receptor from rat cerebral cortex .
● Peptide and peptidomimetic libraries. Molecular diversity and drug design.

15. Split-mix synthesis
Solid support is divided before
each coupling cycle
Equimolar mixture of peptides
Cannot conduct direct mixture
synthesis on solid phase due to
differential reaction rates
One unique peptide on each bead

16. Multi-pin synthesis
The reaction vessel consist of brush like
array of pins, at the end of it consists of
bead with suitable linker (here synthesis
takes place)
Inserted into the plates where the
reagents and the solvents kept and
continuously changed.

17. Solution phase synthesis
Unlike one bead-one compound synthesis, it
lead to mixture of products in one pool.
Most of the reaction occurs in solution
phase.
Problem: removing unwanted impurities at
each step in synthesis

18. Solid phase v/s solution phase
Solid phase
● Large excess of reagent allowed
● Multistep synthesis allowed
● Easy workup isolation
● Reaction Suitable for few substance
● Mix and split possible
● Expensive
Solution phase
● Optimum (unless purification done)
● No linker/cleavage chemistry
● Purification is difficult
● Suitable for any organic reaction
● Unlimited product quantities
● Inexpensive

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. Computer-Assisted Combinatorial Chemistry and
Molecular Design

21. Overview of combinatorial technologies.

22. Diversity of compounds generated by combinatorial
approach

23. Principle of phage libraries
preparation:
Use of biological display libraries for
the isolation of peptide ligands is an
interesting alternative to chemical
libraries.
Biological approach combinatorial library

24. Case study

25. Case study 1: Inhibitors of influenza
endonuclease
Inhibitors of Influenza Virus Polymerase Acidic (PA) Endonuclease: Contemporary
Developments and Perspectives.

26. Case study 1 : Inhibitors of influenza endonuclease
Based on a pharmacophore hypothesis, novel 1-hydroxy-indolin-2-ones were
proposed as inhibitors of influenza endonuclease
A parallel synthesis was developed which allowed to synthesize a library 131
compounds in significant quantities (6-71 mg) and high purities (75 - 99%) within 4
months
From 131 compounds tested 26 had an IC50< 50 μM
From 26 active compounds 10 showed a good antiviral activity in cell cultures

27. Case study 2: Discovery of Innovative Small Molecule
Therapeutics
Rapamycin is a immunosuppressant natural product,
which has two binding sites. It binds to the FKBP domain
and to mTOR (kinase) effector domain.
Besides its immunosuppressant activity the synthetic
analogue torisel shows potent anti-tumor activity and is
used for treatment of renal carcinoma.
Torisel was obtained through a parallel synthesis
approach from rapamycin.
M. Abou-Gharbia, J. Med. Chem. 2009, 52, 2-9

28. Parallel synthesis starting from a natural product-derived
building block

29. Using a parallel synthesis
approach starting from the
natural product rapamycin, the
alcohol group was derivatized
with various different
substituents.
ILS-920, lacked immuno-
suppressant properties and
demonstrated good brain
penetration.

30. Three families of kinases:
● Serine-threonine kinases (S/TKs)
● Tyrosine kinases (TKs)
● Dual function kinases (DFKs)
Involved in cell signaling pathways
Roughly 2000 kinases known in the human genome
Kinases phosphorylate serine, threonine and
tyrosine and are ATP dependent
Case study 3: Kinase inhibitor

31. Case study 3: Kinase inhibitor
Combinatorial Synthesis of 2,9-Substituted Purines
Purine rings, common structural element of a large number
of agonists, antagonists, substrates and effectors that play
key roles in many cellular processes.
Combinatorial libraries of purine derivatives provide
inhibitors of these processes that are useful biological
probes or lead molecules for drug development efforts.

32. Case study 3: Kinase inhibitor

33. Case study 4 : Development of an orexin receptor positive
potentiator
The structure of the peptoid that arose as a primary hit
from a binding screen is shown at the top. The part of
the molecule found to be important for receptor binding
is highlighted in red.
This putative minimal pharmacophore (11), was
synthesized and shown to have slightly better activity
than the peptoid. The part of 11 with a putative
relationship to Almorexant (see box) is shown in blue.
The structure of an improved peptoid antagonist (12)
and a positive allosteric potentiator (13) are also shown.
The moieties on these molecules thought to be related
to the trimethylfluoro-substituted aromatic ring of
Almorexant are depicted in gold.

34. Reference
● David J. Ecker & Stanley T. Crooke. (1995). Combinatorial Drug Discovery: Which Methods Will Produce the Greatest
Value? Bio/Technologyvolume 13, 351–360.
● Stanislav Miertus, Giorgio Fassina,And P. F. Seneci. (2000) Concepts of combinatorial chemistry and combinatorial
technologies. Chem. Listy 94, 1104-1110.
● J. W. Jacobs et al. (Versicor), 40th annual ICAAC conference, Toronto, Canada, september 17-20th, 2000, Poster 2193 and
2194
● Mario Geysen, H., Schoenen, F., Wagner, D., & Wagner, R. (2003). A guide to drug discovery: Combinatorial compound
libraries for drug discovery: an ongoing challenge. Nature Reviews Drug Discovery, 2(3), 222–230.
● Bhattacharyya, S. (2001). Combinatorial Approaches in Anticancer Drug Discovery: Recent Advances in Design and
Synthesis. Current Medicinal Chemistry, 8(12), 1383–1404.
● Thomas Kodadek and Di Cai. (2010) .Chemistry and Biology of Orexin Signaling. Mol Biosyst. 1366–1375.
● M. Sallberg, U. Ruden, L.O. Magnius, E. Norrby, B. Wahren, Immunol Lett., 1991 Sep;30(1):58-69
● C.R. Baeza, A. Unden, FEBS Lett., 1990 Dec 17; 277(1-2):23-5
● F. al-Obeidi, V.J. Hruby, T.K. Sawyer, Mol. Biotechnol., 1998 Jun; 9(3):205-23
● Musonda, C., & Chibale, K. (2004). Application of Combinatorial and Parallel Synthesis Chemistry Methodologies to
Antiparasitic Drug Discovery. Current Medicinal Chemistry, 11(19), 2518–2533.

35. Thank You