The document discusses drug discovery processes, focusing on target identification and validation through various methods including high-throughput screening (HTS), genomics, proteomics, and bioinformatics. It highlights the significance of understanding molecular targets related to diseases, utilizing transgenic animals for efficacy and specificity testing, and employing techniques such as microarrays and antisense technology. Overall, it emphasizes the integration of multiple scientific approaches to improve drug discovery and development outcomes.

1. Presented by : ANAND SAGAR TIWARI
M.Pharm (Second Semester))

2.  Introduction
 Basic concepts
 Role of different tools in Target discovery and
validation
 Role of transgenic animals in target validation

3.  Historically drugs were discovered by identifying
the active ingredient from traditional remedies or
by serendipitous discovery as with penicillin.
 Recently the diseases are being controlled at
molecular and physiological levels.
 After sequencing of human genome, allowed rapid
cloning HTS has been used against isolated
biological targets.
 Hits from these screens are then tested in cells and
then in animals for efficacy.

6.  What is a target ?
 The target is the naturally existing cellular or
molecular structure involved in the pathology of
interest where the drug in development is meant to
act.
 Established targets are those for which there is a
good scientific understanding and how it is
involved in human pathology.
 A target is termed druggable if its activity can be
modulated by a therapeutic (small drug molecule
or biologic).

7.  Has a confirmed role in the pathophysiology of the
disease.
 Target expression is not evenly distributed
throughout.
 Target’s 3D structure is available to assess
druggability.
 Easily assayable using High throughput screening.
 Should possess a promising toxicity profile,
potential adverse effects which are predictable.
 Proposed target has a favourable intellectual
property (IP) status.

8.  Screening and design: The process of finding a
new drug target for a particular disease usually
involves HTS.
 Herein large libraries of chemicals are tested for
their ability to modify the target.
 e.g. If the target is novel GPCR, compounds will
be screened for their ability to inhibit or stimulate
that receptor.
 Important function of HTS is to show the
selectivity of compounds for the chosen target.

9.  What is Target identification ?
 Target identification is the process of identifying the
direct molecular target. e.g. Protein or nucleic acid of a
small molecule.
 It is aimed at finding the efficacy target of a drug or
other xenobiotics.
 The techniques used may be based on principles of
biochemistry, biophysics, genetics or other disciplines.
 A good drug target needs to be relevant to the disease
phenotype and should be amenable to therapeutic
evaluation.
 Target identification is carried out by target
deconvolution or target discovery.

10. From target
deconvolution and
target discovery
one can move to
target validation.
Target
identification
Have a
compound
Target
deconvolution
Want a
compound
Target
discovery

12.  In target based drug discovery biological targets
are already established.
 Target role in the disease process is known.
 Target is then used to create relevant system based
assays and vast compound libraries.
 These are screened in search of a hit– a candidate
drug.

13.  Target-based drug discovery can exploit numerous
approaches including crystallography,
computational modelling, genomics, biochemistry.
 Thus uncovering how a drug interacts with a target
of interest.
 It also enables:
i. Development of structure activity
relationship(SAR)
ii. Development of biomarkers.
iii. Discovery of future therapeutics that act at the
specific target of interest.

14.  Preclinical drug target validation has the aim to
increase confidence in a particular drug target.
 It is the process by means of which the predicted
molecular target of a small molecule is verified.
 While the validation of a drug’s efficacy and
toxicity in animal models is valuable, the ultimate
test is in clinical setting.
 It is broken down into two key steps
i. Reproducibility
ii. Introduce variation to the ligand

17.  It is in contrast to genetics which refers to the
study of individual genes and their roles in
inheritance.
 Genomics aims at the collective characterization
and quantification of all of an organism’s genes,
their interrelations and influence on an organism.
 Genomics also involves the sequencing and
analysis of genomes through high throughput
DNA sequencing and Bioinformatics to analyze
the function of each genome.

18. Genomics has provided applications in many fields
namely;
 Genomic medicine: Next generation genomic
techniques has allowed researchers to drastically
understand the genetic bases of drug response and
disease.
 Synthetic biology and bioengineering
 Conservation genomics: Conservationist can use
the information gathered by genome sequencing in
order to better evaluate genetic factors key to
species conservation such as the genetic diversity
of a population or whether an individual is
heterozygous for a recessive disorder.

21.  Target identification is based upon molecular
information derived from genome sequences and
protein structures.
 Genomics helps in the identity comparison for
nucleic acid sequences.
 For preparing the databases of model organisms.
This helps to screen:
 Rare specific genes
 Virulence genes
 Bacterial membrane translocation proteins.

22.  During preclinical studies the genes linked with
drug metabolism could be genotyped in patients
recruited for phase I trials.
 If efficacy data are collected during phase I trials,
polymorphisms in the drug target gene could be
typed in phase I participants.
 Their linkage with side-effects or variation in drug
response can be assessed.
 The negative results can be further refined during
phase II clinical trials.

23.  Bioinformatics is an interdisciplinary field that
develops methods and software tools for
understanding biological data, especially when the
data is large and complex.
 Such identification is concerned with the better
understanding of the genetic basis of disease.
 Common uses of bioinformatics include the
identification of candidate genes and single
nucleotide polymorphism
 Bioinformatics has been of great importance to
develop fast and accurate target identification and
prediction method for the drug discovery.

25.  The application of bioinformatics cut across all the
processes of drug discovery, thus;
 Reducing the risk off drug failure
 Making it a bit cheaper
 Reduction of time spent in the discovery
 Automates the entire process, reduces human
intervention.

26.  In target identification
 In target validation
 In lead identification
 In lead optimization
 In preclinical testing
 In clinical trials

27.  Proteomics is defined as the large scale study of
proteins.
 Proteome is defined as the entire set of proteins
that is produced or modified by an organism or
system.
 Proteomic approach of target identification
includes finding an unstable protein that causes
undesirable effect and then usage of a molecule to
identify its effect.

28. 1. Protein expression profiling: identification of
proteins in a sample as a function of a particular
state of cell.
2. Protein network mapping : To determine protein
interaction with each other in living organism.
3. Mapping of protein modifications: Task to
identify how and where proteins are modified
post-translationally.
4. Helps to study drug MoA , Disease Biomarkers,
Epigenetics, Spatial localization etc.

29.  Genome analysis does not account for post
translation processes unlike proteomics.
 Thus focus shifted from genomics to proteomics.
 The significance lies in the comparison of cells
from normal tissue with those representing a
disease state.
 This comparison enables the identification of
disease-specific biomarkers used for diagnostic or
prognostic tests.
 At the same time it identifies target proteins that
have the potential for drug targets.

30.  One such approach is comparative proteomics,
based on labelling proteins from normal and
diseased tissue with different fluorescent dyes Cy3
and Cy5, mixing the proteins together and
separating them by isoelectric point and molecular
weight.
 In addition proteomics can analyze biomarkers by
quantifying individual proteins and show the
separation in between one or more protein spots on
a scanned image from two dimensional gel
electrophoresis.
 e.g. Proteomic differences between early and
advanced stages of an illness can be observed.

33. Microarrays
Zinc finger proteins
Antisense technology

34.  A microarray is a laboratory tool used to detect the
expression of thousand gees at the same time.
 The core principle of microarray is hybridization.
 Each known gene or probe occupies a specific site on
the chip and varying level of fluorescent activity shows
varying level of gene activity.
 A DNA microarray also known as DNA chip or biochip
is a collection of microscopic DNA spots attached to a
solid support surface.

36.  The microarray technique has been sub-classified
based on the sample to be analysed:
 DNA microarray
 Protein microarray
 Antibody microarray
 Tissue microarray
 Chemical compound microarray

37.  Microarray based studies provide essential impetus for
identification of disease causing genes in malignancies.
 Has a role in identification of genes for new and
unique potential drug targets.
 It also predicts drug responsiveness for individual
patients.
 It helps to finally initiate gene therapy and prevention
strategies.

38.  Array based gene expression analysis has enabled
parallel monitoring of cellular transcription at the
level of the genome.
 Understanding of normal and abnormal cell
biochemistry and thus on the choice of targets for
drug design.
 e.g. Data generated from High density
oligonucleotides microarray in oncology identified
97 genes as physiological targets.
 Further characterization of these genes should
provide insights into proliferation pathway thus
providing potential therapeutic targets.

40.  A protein microarray is a high throughput method
used to track the interactions and activities of
proteins.
 It also helps to determine their function on a large
scale.
 Since most of the drug targets are proteins, these
microarrays are set to have an important impact on
drug discovery.
 Profiling the different expression of protein using
antibody arrays and correlation of the same to a
disease, potential targets for a disease can be
identified.

41.  Protein microarrays require less sample
consumption and have potential for
miniaturization.
 It provides direct information on protein function
and potential drug targets by studying protein-
protein, protein-lipid, protein-DNA interaction.
 Proteins can be immobilised by :
 Surface modification
 Using expressed protein ligation
 Using Click chemistry etc.

44.  Antisense oligonucleotides are short , single
stranded DNA molecules that interact with mRNA
to prevent translation of a targeted gene.
 This is done by preventing the mRNA instructions
from reaching the ribosome and inhibiting protein
synthesis.
 Thus antisense oligonucleotides are unmodified or
chemically modified ssDNA, RNA or their
analogs.
 Two classes can be discerned i.e. RNase H
dependent oligonucleotides and steric blocker
oligonucleotides.

45.  For successful application of antisense
oligonucleotides several problems need to be
addressed. They are:
 Accessible sites of target RNA for oligonucleotides
binding have to be identified.
 They need to be protected against nuclease enzyme
attack.
 Cellular uptake and correct intracellular
localization should be made feasible.
 E.g. Phospho-thioate-deoxy-nucleotides, 2-O-
methyl etc.

46.  Small interfering RNA or short interfering RNA
are 20-25 base pair in length.
 It interferes with the expression of specific genes
with complementary nucleotide sequence.
 Thus degrading mRNA after transcription,
preventing translation.
 The Dicer enzyme catalyzes production of siRNAs
from long dsRNAs and small hairpin RNAs.

47.  Artificial induction of the natural RNAi is now
routinely used to specifically silence target genes.
 Applied for gene function, identification of
molecules involved in biochemical pathways, lead
optimisation and functional genomics.
 The most common use is in target validation
experiments.
 They are able to regulate the expression of genes
by a phenomenon known as RNA interference.

51.  A zinc finger is a small protein structural motif.
 These are among the most abundant proteins in
eukaryotic genomes.
 They have a diverse functions including DNA
recognition, RNA packaging, transcriptional
activation, regulation of apoptosis, protein folding.
 Zinc finger proteins that recognise novel DNA
sequences form the basis of drug discovery and
therapeutics.

52.  These proteins have been used as the DNA-
binding domains of novel transcription factors.
 Useful for validating genes as drug targets.
 Also used for engineering cell lines for small
molecule screening and protein production.
 Recently these are used as a basis for novel human
therapeutics.
 Direct way involves designing ZFPs to validate the
phenotypic effects of activating or repressing a
gene.
 Alternatively libraries may be used to screen cells
for desired phenotypic changes.

53.  The use and usefulness of transgenic animals in
the discovery of new medicine has been vital these
days.
 Mouse and man are very similar at the genetic
level.
 Genetic alteration in the mouse often result in
functional changes predicting the relevant
pharmacological effect in man.
 Genetically modified mice possess many
characteristics making them useful for
understanding novel disease mechanism.

54. Use of transgenic in
drug discovery
Target testing
Efficacy
testing
Specificity
testing
Compound
testing
Chemistry
related
toxicology

55.  Transgenic animals can be used widely in the
process of discovering new medicines, e.g. In
validation of potential new drug targets and in
understanding of specificity, efficacy & safety of
compounds. Other roles are:
 Disease modelling and drug development
 Target validation
 Compound validation
 Efficacy evaluation
 Specificity evaluation
 Safety evaluation
 Replacing non-human primate models