Target Validation Introduction,Drug discovery, Target identification and validation, Target validation and techniques By Ms. B. Mary Vishali Department of Pharmacology

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
TARGET VALIDATION
A Seminar as a part of curricular requirement
for I year M. Pharm II semester
Presented by
Ms. B. Mary Vishali
(Reg. No. 20L81S0104)
Department of Pharmacology
Under the guidance/Mentorship of
Dr. P. Ramalingam., Ph.D.
Director- R&D Division,
Professor of Pharmaceutical Analysis
and Medicinal Chemistry.

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction
• Drug discovery
• Target identification and validation
• Target validation and techniques
• References
CONTENTS:

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Introduction
oA drug discovery program initiates because there is a disease or
clinical condition without suitable medical products available
oThe aim is to achieve registration by one or more regulatory
authorities, to allow the drug to be marketed legally as a medicine for
human use.
oDeveloping a new drug is a complex process which can take 12–15
years and cost in excess of $1 billion.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Drug Discovery and Development:

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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5

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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Target identification and validation
oOne of the most important steps in developing a new drug is target
identification and validation.
oA target is a broad term which can be applied to a range of biological
entities which may include for example proteins, genes and RNA.
oA good target needs to be efficacious, safe, meet clinical and
commercial needs and, above all, be ‘druggable’.
oEx: Proton pump (H+K +ATPase) of parietal cells of stomach

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Strategies used in target discovery
Two main strategies are employed;
1. Molecular approach: Target discovery through understanding of
cellular mechanisms (ex: Tissues, cell lines)
2. Systems approach: Target discovery through the study of disease in
whole organism (Clinical sciences, patient/animal models)

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Target validation
o Once a gene target or a mechanistic pathway is identified, the next
step is to demonstrate that it does play a critical role in disease
initiation, perpetuation, or both.
o The role of target validation, therefore, is to demonstrate the
functional role of the potential target in the disease phenotype.
o The target validation normally require that the;
Target is expressed in the disease-relevant cells/tissues, and
Target modulation in cell and/or animal models ameliorates the
relevant disease phenotype

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1. mRNA modulation (antisense strategies)
Oligonucleotides
 ribozymes and small interfering RNA (siRNA)
2. Zinc finger printing
3. Transgenic animals
Techniques used in target validation

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Antisense Technologies
Antisense technology is a potentially
powerful technique which utilizes a strand
of nucleic acid (DNA, RNA or a chemical
analogue) which are designed to be
complimentary to a region of a target
mRNA molecule.
Binding of the antisense oligonucleotide to
the target mRNA results in its inactivation
and hence, effectively turning off the gene.

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Types of antisense strategies
Antisense oligonucleotides
They usually consist of 15– 20 nucleotides, which are complementary
to their target mRNA. They block translation of the mRNA or induce
its degradation by RNase H

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1st Generation 2nd Generation 3rd Generation
Here one of the
nonbridging oxygen
atoms in the
phosphodiester bond is
replaced by sulphur.
The modification
improves;
• nuclease resistance,
• Half life (from 1h to
10hrs),
• activate RNase H,
• 2’-O-methyl and 2’-
O-methoxy-ethyl
RNA are the most
important members of
this class.
• They are less toxic
than phosphorothioate
DNAs and have a
slightly enhanced
affinity towards their
complementary RNAs
DNA and RNA
analogues with modified
phosphate linkages or
riboses as well as
nucleotides with a
completely different
chemical moiety
substituting the furanose
ring (5 membered ring)
have been developed

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Ribozymes (RNA enzymes) and Small interfering RNA
(siRNA)
o Ribozymes are unique RNA enzymes that can recognize complementary
mRNA targets and cleave them at “GUC” sites in a sequence-specific
fashion.
o This makes ribozymes powerful tools for studying the functional
consequences of suppressed gene expression.
o RNAi is a natural process that occurs in many organisms ranging from
plants to mammals.
o In this process, double-stranded RNA or hairpin RNA is cleaved by a Rnase
IIItype enzyme called Dicer into small interfering RNA duplex.
o This then directs sequence-specific, homology-dependent,
posttranscriptional gene silencing by binding to its complementary RNA
and triggering its elimination through degradation or by inducing
translational inhibition.

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Zinc Finger Proteins (ZFP)
o Zinc fingers are small protein domains in which zinc plays a
structural role contributing to the stability of the domain.
oBased on the structural properties in the vicinity of the zinc‐binding
site they are grouped into 8 fold groups.
1. C2H2 ‐like finger
2. Gag knuckle
3. Treble clef finger
4. Zinc ribbon
5. Zn2/Cys6‐like finger
6. TAZ2 domain‐like
7. Short zinc‐binding loops
8. Metallothioneins

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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• Proteins bind zinc as a cofactor for catalysis or as a structural
stabilizer.
• In zinc fingers, the role of zinc is structural and zinc ions typically do
not participate in the function directly.
• Other parts of a zinc‐binding molecule bear functional importance. •
Small protein domains assembled around zinc ions are versatile
structural templates that perform various functions.
• They are involved in;
 nucleic acid (DNA and RNA) binding,
 protein–protein interactions,
 binding small ligands (lipids) and
 sometimes also possess enzymatic properties
Functional properties of ZFP

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• Transgenic technology represents an attractive approach to
• reduce the attrition rate of compounds entering clinical trials
• increases the quality of the target and compound combinations making the transition from
discovery into development.
• Transgenic technology influences decision making in target identification, target validation,
and can also provide better models for human diseases, Also as model designed to alert
researchers early about potential issues with drug metabolism and toxicity.
• There is a need for in vivo target validation data before large amounts of resource are
invested in the potential target. • Considerable efforts are being made to identify rapid,
reliable and general tools for in vivo validation, but, so far, only transgenic animals work
reliably on a wide range of targets. • A common requirement in the target validation phase is
the production of genetically modified animals that either overexpress (gene addition) or no
longer possess the target (knockout animals). • These animals provide in vivo functional data
on a potential target which are often lacking.
Transgenic animals in drug discovery

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Alzheimer’s disease
• No animal models existed for the disease before transgenic
technology was employed.
• Today, several transgenic models have been established. These
models resemble much of the human pathology and are frequently
used in the search for new therapeutic opportunities.
• Immunisation of Amyloid precursor protein (APP) transgenic mice
with the protein Aβ42 before disease became established resulted in
disease prevention and, if performed on older animals, inhibited
disease progression.
• This study using transgenic mice suggests that vaccination against
Alzheimer’s disease could have potential as a therapeutic approach.

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1. Boscha, F., Rosich, L. (2008), The Contributions of Paul Ehrlich to
Pharmacology, 171-179.
2. Terstappen, G., Schlupen, C., Raggiaschi, R., & Gaviraghi,
G.(2007), Target deconvolution strategies in drug discovery, 891-
903.
3. Choe, g., Horvath, S., Cloughesy, T. F., et al. (2003) Analysis of the
phosphatidylinositol 3-kinase signaling pathway in gliblastoma
patients in vivo. Cancer Res. 63(11), 2742-2746.
REFERENCES :

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