PRINCIPLES OF DRUG DISCOVERY (MPL 203T)

Seminar on:

LEAD IDENTIFICATION
PRESENTED BY: DEVESH YADUVANSHI
M.PHARM. 2nd SEM. (2025)
DEPARTMENT OF PHARMACOLOGY
SPER, JAMIA HAMDARD, NEW DELHI
 CONTENTS

S. No. Topic Page No.

1. INTRODUCTION 1-3

METHODS OF LEAD IDENTIFICATION:

• Combinatorial chemistry
2. • High-throughput screening 4-23
• In silico lead discovery techniques
• Assay development for HIT identification

5. REFERENCES 24
 INTRODUCTION

• A lead compound is generally defined as, a new chemical entity that could potentially be
developed into a new drug by optimizing its beneficial effects and minimizing its side effects.

• Once a target & a testing system has been chosen, the next step is to find a lead compound which
shows the desired pharmaceutical activity.

• The identification of small molecule modulators of protein function and the process of
transforming these into high content lead series are key activities in modern drug discovery.

• A lead compound is a first foothold on the drug discovery ladder.

• It takes much more effort to make a lead compound into a drug candidate.

1
 FINDING LEAD COMPOUND
Possible routes to identifying a lead compound
are:
1. Chance observations.
2. Selective optimization of side activities
(SOSA approach).
3. Herbal folk remedies.
4. Screening of natural product metabolites.
5. Screening of compound libraries (High-
throughput).
6. "Rational" drug design.
7. Natural substrate-based drug design, etc.

2
 SCREENING IN LAB.

• Without some reliable measure of potency, it is impossible to conduct a systematic analysis of

possible new drugs.

• Screening or assaying

• “The testing of a (series of) molecule against a known biological target that correlates with a cellular
or pharmacological activity is known as screening”, (e.g. enzyme inhibition or receptor binding)

• This is now made possible/easier in modern research because macro-molecular targets, (e.g. proteins,
enzymes/receptors) can be identified.

• Targets are now available in large quantities using molecular biology.

• Automated (High-throughput screening) technologies available.

3
 METHODS OF LEAD IDENTIFICATION

HTS (High-
Combinatorial In-silico lead Assay development
throughput
chemistry discovery techniques for HIT identification
screening)

4
 COMBINATORIAL CHEMISTRY
• Combinatorial chemistry is a technique by which large numbers of different but structurally
similar molecules are produced rapidly and submitted for pharmacological assay.

• This technique uses the same reaction conditions with the same reaction vessels to produce a large
range of analogues.

COMBINATORIAL CHEMISTRY WITHIN DESIGN

Therapeutic Lead Lead Development

Drug
target discovery optimization candidate

COMBINATORIAL CHEMISTRY
CAN IMPACT HERE
5
 SYNTHETIC METHODOLOGIES FOR PRODUCTION
OF LIBRARIES
1. Solid Phase Techniques.

2. Parallel Synthesis:
i) Houghton's Tea Bag Procedure.
ii) Automated parallel synthesis.
iii) Automated parallel synthesis of all 27 tripeptides from 3 amino acids.

3. Mixed Combinatorial Synthesis.

4. Solution phase synthesis.

6
 SOLID PHASE TECHNIQUE
• Reactants are bound to a polymeric surface and modified whilst still attached.
• Final product is released at the end of the synthesis.
 Requirements:
• A resin bead or a functionalized 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.
• A means of cleaving the product from the linker at the end.
• Protecting groups for functional groups not involved in the synthesis.
 Procedure:
• Beads must be able to swell in the solvent used, and remain stable most reactions occur in the bead
interior.
7
 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.

8
 PARALLEL SYNTHESIS
 Aim: 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 optimization.

1. 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 synthesized 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 quantities of different products.
9
 2. Automated Parallel Synthesis:
• Automated synthesizers are available with 42, 96 or 144 reaction vessels or wells.
• Use beads or pins for solid phase support.
• Reactions and workups are carried out automatically.
• Same synthetic route used for each vessel, but different reagents.
• Different product obtained per vessel.

3. 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. 10
 HIGH THROUGHPUT SCREENING
• High Throughput Screening (HTS) is a drug discovery process widely used in pharmaceutical
industry. It leverages automation to quickly assay the biological activity of a large number of drug
like compound.
• HTS rapidly tests thousands to millions of chemical compounds against biological targets using
robotics and automation.
• It can be used for target-specific interactions (enzyme/receptor binding) or broader
cellular/phenotypic responses.
• Uses microplates (e.g., 96, 384, or 1536 wells) to conduct multiple tests simultaneously, reducing
reagent costs.
• Generates massive datasets, analyzed using bioinformatics and AI to identify potential lead
compounds.
• Requires robust, reproducible biochemical or cell-based assays to ensure meaningful results.
• Helps identify "hit" compounds, which can be further optimized into drug candidates.

11
TYPES OF HTS ASSAY IN DRUG DISCOVERY

Assays in drug discovery

Biochemical assays Cell-based assays

Phenotype-based:
Target-based: 1. Transcriptional read-outs
Enzymes (e.g., kinases), 2. Second messenger level
Receptors (e.g., Nuclear 3. Protein interactions
receptors) 4. Cell viability
12
TARGETS IN HTS

13
DETECTION METHODS IN HTS USES OF HTS
• Spectroscopy (e.g., Fluorescence spect.,  To screen for all kind of novel biological
NMR, Total internal reflection fluorescence, active compounds:
light scattering) 1. Natural products.
• Chromatography (e.g., GC, TLC & HPLC.
2. Combinatorial libraries.
• Mass spectroscopy.
3. Biological libraries.
• Calorimetry.  To screen Micro arrays such as:
• X-ray diffraction (crystallography).
1. DNA chips.
• Microscopy.
2. RNA chips.
• Radioactive methods.
3. Protein chips.
ADVANTAGES OF HTS
• High sensitivity of assay.
• High speed of assay.
• Low background signal.
14
 IN SILICO LEAD DISCOVERY TECHNIQUE
In silico is an expression used to mean performed on computer or via simulation. In silico drug
designing is defined as the identification of the drug target molecule by employing bioinformatics tools.
It includes:
• Virtual Screening (VS): Uses computational methods to screen large compound libraries against a
biological target, predicting potential drug candidates.
• Molecular Docking: Simulates interactions between small molecules (ligands) and a target protein to
predict binding affinity and stability.
• Pharmacophore Modeling: Identifies key molecular features (such as hydrogen bond donors,
acceptors, hydrophobic regions, and aromatic rings) essential for biological activity, aiding in the design
of new drug candidates.
• Quantitative Structure-Activity Relationship (QSAR): Develops mathematical models correlating
chemical structure with biological activity, helping predict the potency of novel compounds.
• Molecular Dynamics (MD) Simulations: Studies the behavior of molecules over time to assess
stability, flexibility, and binding mechanisms.
• De Novo Drug Design: Constructs new drug-like molecules from chemical spaces using computational
approaches based on target-specific interactions.
• Artificial Intelligence (AI) and Machine Learning (ML) in Drug Discovery: Enhances lead
15
 TYPES OF IN SILICO DRUG DESIGNING

In silico
Ligand based Structure based
drug designing drug drug designing
designing

16
 LIGAND-BASED DRUG DESIGN
• Ligand-based drug design (LBDD) focuses on designing new drugs based on the knowledge of
existing ligands that bind to a target protein. Used to derive a Pharmacophore.

• It relies on the assumption that molecules with similar structures will exhibit similar biological
activities.

• Includes quantitative structure-activity relationship (QSAR) modeling, pharmacophore modeling, and

similarity searching to predict and optimize drug candidates.

• Requires a set of known active compounds to derive molecular patterns, descriptors, and properties
that correlate with biological activity.

• Applications: Used when the 3D structure of the target protein is unknown, making it useful for de
novo drug discovery and lead optimization.

• Advantages: Faster and cost-effective compared to structure-based drug design, as it can predict
17
potential drug candidates without needing detailed structural information of the target.
 STRUCTURE-BASED DRUG DESIGN
Structure based drug design (SBDD) relies on knowledge of the 3-D structure of the biological
target obtained through methods such as:
• X-ray crystallography.
• NMR spectroscopy.
• Homology modelling.
Using the structure of the biological target, drugs that are predicted to bind with to the target may be
designed using:
• Interactive graphics.
• The intuition of a medicinal chemist.
• Automated computational procedures.
 Disease selection and involved pathway identification.
 Target selection (e.g., enzymes, receptors or nucleic acid).
 Target validation: From RCSB (The Research Collaboration for Structural Bioinformatics) PDB
(Protein Data Bank).
18
19
 MOLECULAR DOCKING
• In the field of molecular modeling, docking is a method
which predicts the preferred orientation of one molecule to
a second when bound to each other to form a stable
complex.

• Knowledge of the preferred orientation in turn may be

used to predict the strength of association or binding
affinity between two molecules using, for example, scoring
functions.

20
 HIT IDENTIFICATION
 Assay Development for HIT identification:

• A hit is a compound which has the desired activity in a compound screen and whose activity is
confirmed upon retesting.

• Lead compounds are chemical compounds that show desired biological & pharmacological
activity and may initiate the development of a new clinically relevant compound.

 Hit identification:

• For Hit identification, some information about either the target protein or an active compound is
necessary.

• If the structure of the natural substrate is known, a ligand-based approach will be accomplished.

21
 PROCESS OF HIT IDENTIFICATION
Compound libraries Target identification

Target validation

Assay development

HTS

Lead optimization

Clinical trial
22
 BIOCHEMICAL ASSAYS
• Measure function of a purified target.
• Identify compounds that modulate the activity of the target protein.
• Recombinant proteins, proteins isolated from crude cell lysates.
• Examples: Kinase/ATPase assays, Protease assays & Protein interaction assays.

CELL-BASED ASSAYS
• Provide a functional read out of compound activity.
• Useful for follow up of biochemical assays.
• Examples: Cell proliferation: MTT etc, for oncology.
Apoptosis: second messenger levels.
Motility/Migration: Bacterial viability assays.

23
 REFERENCES
1. Gandhi, P., Clinical research methodology. Indian Journal of Pharmaceutical Education and
Research, Vol. 45(2), 2011. pp. 199-209.
2. Graham L. Patrick. Combinatorial and parallel synthesis, An introduction to Medicinal
Chemistry, 5th edition. Oxford, U.K: Oxford University Press; 2012. pg. 313-379.
3. Rao, V.S. and Srinivas, K., Modern drug discovery process: An in silico approach. Journal of
bioinformatics and sequence analysis, Vol. 2(5). 2011. pp.89-94.
4. Gopi, C., Krupamai, G., Sri, C.S. et al. An overview of recent progress in modern synthetic
approach—combinatorial synthesis. Beni-Suef Univ J Basic Appl Sci 9, 57 (2020).
https://doi.org/10.1186/s43088-020-00083-7
5. Ravi Kumar. Drug Discovery (Lead Identification and High Throughput Screening). Research
Journal of Pharmacology and Pharmacodynamics. 2021; 13(2):46-0. doi:10.52711/2321-
5836.2021.00010.
6. Other internet sources.
24
27