Target and Lead Identification

Drug discovery and development

Process usually starts with choosing a disease

 Drug design and development
Stages
1) Identify target disease
2) Identify drug target
3) Establish testing procedures
4) Find a lead compound
5) Structure Activity Relationships (SAR)
6) Identify a pharmacophore
7) Drug design- optimising target interactions
8) Drug design - optimising pharmacokinetic properties
9) Toxicological and safety tests
10) Chemical development and production
11) Patenting and regulatory affairs
12) Clinical trials
 Target disease
Priority for the Pharmaceutical Industry
• Can the profits from marketing a new drug outweigh the cost
of developing and testing that drug?

Questions to be addressed
• Is the disease widespread?
(e.g. cardiovascular disease, ulcers, malaria)
• Does the disease affect the first world?
(e.g. cardiovascular disease, ulcers)
• Are there drugs already on the market?
• If so, what are their advantages and disadvantages?
(e.g. side effects)
• Can one identify a market advantage for a new therapy?
 Drug targets
A) LIPIDS
Cell Membrane Lipids

B) PROTEINS
Receptors
Enzymes
Carrier Proteins
Structural Proteins (tubulin)

C) NUCLEIC ACIDS
DNA
RNA

D) CARBOHYDRATES
Cell surface carbohydrates
Antigens and recognition molecules
 Drug targets
TARGET SELECTIVITY

Between species
• Antibacterial and antiviral agents
• Identify targets which are unique to the invading pathogen
• Identify targets which are shared but which are significantly different in
structure

Within the body

• Selectivity between different enzymes, receptors etc.
• Selectivity between receptor types and subtypes
• Selectivity between isozymes
• Organ selectivity
 Human genome project

Aims of the project:

• An international research effort to determine the sequence of the
human genome and identify all the genes that it contains.
• To understand the function of the genes
 Milestones
1986 Human Genome Project began
1990 Project initiated as joint effort of US Department of
Energy and the National Institute of Health.
1994 Genetic Privacy Act: to regulate collection, analysis,
storage and use of DNA samples and genetic information
is proposed.
1996 Welcome Trust joins the project.
1998 Celera Genomics (a private company founded by Craig Venter)
formed to sequence much of the human genome in 3 years.
1999 Completion of the sequence of Chromosome 22-the first
human chromosome to be sequenced.
2000 Completion of the working draft of the entire human genome.
2003 HGP sequencing is completed and Project is declared finished
two years ahead of schedule.
 Revelations from the HGP

• The human genome is nearly the same (99.9%) in all people

• Only about 2% of the human genome contains genes, which are
the instructions for making proteins
• Humans have an estimated 30,000 genes
• The functions are unknown for over 50% of the discovered
genes.
• Approved small molecule drugs target approx. 200 human protein
targets
• Many more suitable protein drug targets remain to be discovered
 Impact of the human genome project
The Past
Active Compound Target

The Present/Future

Target Active compounds

Potential benefits
• Improved diagnosis of disease
• Detect genetic predispositions to disease
• Design “custom drugs” (pharmacogenomics) based on individual genetic
profiles
https://www.technologynetworks.com/drug-discovery/articles/target-identification-validation-in-drug-discovery-312290
 Target-based vs Phenotypic screening
Target-based screening:
Activity of compounds are examined at targets known or postulated to be
involved in a disease.

Advantages:
1. Generally faster, more economical
2. Ability to evaluate the drug in the absence of other complicating factors
(e.g. pharmacokinetics)
3. May lead to detailed knowledge of how the drug interacts with the target

Phenotypic screening:
Activity of compound are examined in cells, tissues or animals.

Advantages are:
1. Does not require knowledge of the target
2. More representative of actual clinical situation
3. Able to identify pro-drugs
4. Can be used to access less obvious targets – e.g. a previously unknown
enzyme or receptor
 Target deconvolution and Target discovery

Target deconvolution
• an active compound is known but the target is not known
• active compound is often derived from a phenotypic screen

Target discovery
• goal is to find a target that can be used as a potential target for target-
based screening
• starting point is different - no compound
Target deconvolution by affinity chromatography

• Most widely used method

• Structure-activity relationship studies are needed
• Synthesis sometimes difficult with natural products
• Compounds can lose biological activity
• Membrane-bound targets difficult to identify by this method
 Target deconvolution by DARTS

• DARTS – Drug affinity responsive target stability

• Binding of drug stabilizes protein (can mask protease recognition sites)
• Target for resveratrol identified as eIF4A (eukaryotic translation initiation
factor 4A)
Target deconvolution by profiling
 Target identification by genetic methods

1. Down-regulating or over expression of specific genes in

model organisms (e.g. down-regulation of gene in cancer cell
leads to lack of growth of cancer cell implies that the gene-
product might be a good drug target).

2. Comparison of genes/proteins in healthy and diseased

individuals
 Target validation

Goal: Establish that engaging the target produces meaningful

therapeutic benefit
May involve :
1. Screening compounds at target (reproducibility is important)
2. Determining target engagement in vivo
3. Evaluating effects in vivo

Genetic knockouts can also be used for target validation

 In vitro Tests
• Tests not carried out on animals/humans
Target molecules (e.g. isolated enzymes or receptors)
Cells
Tissues (e.g. muscle tissue)
Organs
Micro-organisms (for antibacterial agents)
• More suitable for routine testing
• Used in high throughput screening
• Measure the interaction of a drug with the target but not the
ability of the drug to reach the target
• Results are easier to rationalise - less factors involved

Drawbacks
• Does not demonstrate a physiological or clinical effect
• Does not identify possible side effects
• Does not identify effective prodrugs
 In vivo Tests
• Carried out on live animals or humans
• Measure an observed physiological effect
• Measure a drug’s ability to interact with its target and its
ability to reach that target
• Can identify possible side effects
• Rationalisation may be difficult due to the number of factors
involved
• Transgenic animals - genetically modified animals
• Drug potency - concentration of drug required to produce 50%
of the maximum possible effect
• Therapeutic ratio/index - compares the dose level of a drug
required to produce a desired effect in 50% of the test sample
(ED50) versus the dose level that is lethal to 50% of the sample
(LD50)
 High throughput screening (HTS)
• A heavily automated procedure involving the use of
robotics and miniaturization of in vitro tests
• Several thousand compounds can be tested at once
• Promiscuous hits and false positives can be a problem
(possibly by aggregation)
 Enzyme Inhibition Tests

• Identify competitive or non competitive inhibition

• Strength of inhibition measured as IC50

• IC50 = concentration of inhibitor required to reduce enzyme

activity by 50%
 Testing with Receptors

• Not easy to isolate membrane bound receptors

• Carried out on whole cells, tissue cultures, or isolated organs

• Affinity - strength with which compounds bind to a receptor

• Efficacy - measure of maximum biochemical effect resulting from

binding of a compound to a receptor.

• Potency - concentration of an agonist required to produce 50%

of the maximum possible effect.
 The Lead Compound
Introduction
• A compound demonstrating a property likely to be
therapeutically useful
• The level of activity and target selectivity are not crucial
• Used as the starting point for drug design and development
• Found by design (molecular modelling or NMR) or by screening
compounds (natural or synthetic)
• Need to identify a suitable test in order to find a lead compound
• Active Principle - a compound that is isolated from a natural
extract and which is principally responsible for the extract’s
pharmacological activity. Often used as a lead compound.
 Sources of Lead Compounds
Via screening from various sources:

Plantlife (flowers, trees, bushes)

Micro-organisms (bacteria, fungi)
A) The Natural World/Medical Animal life (frogs, snakes, scorpions)
Folklore/Herbal Medicine Biochemicals (Neurotransmitters, hormones)
Marine chemistry (corals, bacteria, fish etc)

B) The Synthetic World Chemical synthesis (traditional)

Combinatorial synthesis
Fragment based

C) The Virtual World Computer aided drug design

Virtual screening
 Other Sources of Lead Compounds
D) Improvement of existing drugs
- “me too” and “me better” drugs
 Other Sources of Lead Compounds
- enhancing a side-effect (SOSA approach)

- drug repurposing

• Initially marketed as sedative-hypnotic

• Withdrawn due to teratogenic effects
• New use as immunomodulator
 Other Sources of Lead Compounds

E) Serendipity
 Lead Compounds from the Natural World
A) Isolation and purification
solvent-solvent extraction
chromatography
crystallisation
distillation

B) Structure determination
elemental analysis
molecular weight
mass spectrum
infra red
ultra violet
nmr (1H, 13C)
X-ray crystallography
 Isolation of Natural Products
1. Extract plant material with solvent (eg. Hexanes, acetone, water)

2. Remove solvent
Isolation of Natural Products
3. Test extract

Bioassay – eg. Enzyme, receptor, microbe

 Isolation of Natural Products
4. Chromatography to separate fractions/components

Steps 3 and 4 can be iterative

- bioassay-guided isolation
 Lead Compounds from the Natural World
PLANT EXTRACTS

MORPHINE

POPPY CAPSULE
Extraction Process

Patented method - United States Patent

6,054,584
 Lead Compounds from the Natural World
PLANT EXTRACTS

GALANTAMINE
 Natural product derivatives
PLANT EXTRACTS

WILLOW TREE - SALICYLIC ACID

O OH O OH
Acetic
OH anhydride O CH3
Aspirin
O

COCA BUSH - COCAINE

Me
CH3
N
CO2Me

H
N Procaine
O
O CH3 C NH2
H C O
O
 Lead Compounds from the Natural World
O
VENOMS AND TOXINS C OH
O
C N
Teprotide O O O
H
C N CH C N CH C N
O H
O CH2 CH CH3
C N CH C N CH2 CH2
O O H
CH2 C O CH3
H2N CH C N CH C N
H CH2 NH2
CH2 CH2 O
CH2
CH2 C OH
NH O
C O
HN C NH C N
OH
NH2
CH3
HS

Captopril
(anti-hypertensive)
 Lead Compounds from the Natural World

VENOMS AND TOXINS

MeO
CH 3
N
HO CH 3
O H

H O
H3 C
N OH
H3 C
OMe

Tubocurarine
(from curare)
 Lead Compounds from the Natural World
MeO
VENOMS AND TOXINS CH 3
N
HO CH 3
O H

H O
H3 C
N OH
H3 C
OMe

Tubocurarine
(from curare)

MeO OMe
O O
CH 3 H
N C C N
MeO O (CH 2)5 O OMe

OMe MeO
OMe OMe

Atracurium
(Neuromuscular blocker)
Lead Compounds from the Natural World

MARINE NATURAL PRODUCTS

Lead Compounds from the Natural World

ANIMAL SOURCES

EPIBATIDINE
 Lead Compounds from the Natural World
ENDOGENOUS COMPOUNDS

NATURAL LIGANDS FOR RECEPTORS

OH OH
H
H
N
N
HO HO
Me Agonist
HO
HO
ADRENALINE SALBUTAM OL

NH2 NMe2

HO MeHN
S Agonist
O O
N N
H H
5-HYDROXYTRYPTAM INE SUM ATRIPTAN
 Lead Compounds from the Natural World
ENDOGENOUS COMPOUNDS

NATURAL LIGANDS FOR RECEPTORS

OH O N
H
Antagonist
H
HO N OH
Me

HO
ADRENALINE PROPRANOLOL

Me
NH2 H
N NHMe
HN S
HN
N
N
HISTAM INE
CN Antagonist
CIM ETIDINE
 Lead Compounds from the Natural World
ENDOGENOUS COMPOUNDS

NATURAL SUBSTRATES FOR ENZYMES

Enkephalins Enkephalinase inhibitors

Peptides Protease inhibitors

H CO2H O
N O
H H H H N
N N H
N N
H2N H H H
H O OH
O CONH2
H
L-Phe-L-Pro
SAQUINAVIR
Lead Compounds from the Synthetic World

O
H2N N N S NH2
PRONTOSIL
O
NH2
Lead Compounds from the Synthetic World
O
H 2N S NH2
O

SULFANILAMIDE
Lead Compounds from the Synthetic World
Organic Chemistry

Several compounds synthesized and

tested
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS

AUTOMATED SYNTHETIC MACHINES

Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - PEPTIDE SYNTHESIS

AMINO ACID

RESIN
BEAD
AMINO ACIDS

PEPTIDE
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - HETEROCYCLIC SYNTHESIS

RESIN
BEAD

N
N
Y

CHR1R2
O
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - HETEROCYCLIC SYNTHESIS

N
N
RESIN
BEAD
R2
R3
O
H
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - HETEROCYCLIC SYNTHESIS

N
N
RESIN
BEAD
R2
R3
O
R4
EtO R5
O
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - HETEROCYCLIC SYNTHESIS

N
N
RESIN
BEAD
R2
R3
N
R4
HN R5
O
Lead Compounds from the Synthetic World
COMBINATORIAL SYNTHESIS - HETEROCYCLIC SYNTHESIS

RESIN
N BEAD
N
Y
R2
R3
N
R4
HN R5
O
Lead Compounds – Computer aided drug design

• Design compounds to fit target (intermolecular forces important)

• Use target to virtually screen database of compounds to identify lead
Fragment-based lead discovery

NMR SPECTROSCOPY
Fragment-based lead discovery

Binding Site

Protein
 Fragment-based lead discovery

Protein

NO OBSERVABLE BIOLOGICAL EFFECT

 Fragment-based lead discovery

CH3
CH CH
CH3
CH2

CH2

C CH
C

13C NMR
 Fragment-based lead discovery

CH3
CH CH
CH3
CH2

CH2

C CH
C

13C NMR
Fragment-based lead discovery

Protein

Optimise
epitope
Fragment-based lead discovery

Protein

Optimise Optimise
epitope epitope
Fragment-based lead discovery

Link

Protein

Optimise Optimise
epitope epitope
Fragment-based lead discovery

LEAD COMPOUND
Fragment merging, linking, growing approaches
Biochemistry 2012, 51, 4990−5003
 Fragment-based lead discovery

Design of a lead compound as an immunosuppressant

OH
O

OMe N
H
N O HO
O
O Epitope B

MeO OMe
OMe

Epitope A
 Fragment-based lead discovery
Design of a lead compound as an immunosuppressant

OH
O

O N
H
N O HO
O
O

MeO OMe
OMe

Lead compound
Zelforab – first clinically approved drug based of FBDD (2011)

Approved for metastatic melanoma