drug targets and target identification

1. Lecture 8- Drug Targets and Target identification
BTT- 516– Drug Designing and Development

2. Topic To be covered
1. Drug targets and their type
2. Introduction to drug targets
-Different targets
-Nature of targets
3. Homework

3. Introduction
Identifying the biological origin of the disease and the potential
targets for involvement, is the first step in the discovery of a
medicine.
Target identification is process of identifying the direct
molecular target (protein, nucleic acid, or small molecule).

4. Biological Targets
 A biological target is anything within a living organism to which some
other entity (like an endogenous ligand or a drug) is directed and/or
binds, resulting in a change in its behavior or function
 Drug target: The term "biological target" is frequently used
in pharmaceutical research to describe the native protein in the body
whose activity is modified by a drug resulting in a specific effect, which
may be a desirable therapeutic effector an unwanted adverse effect. In
this context, the biological target is often referred to as a drug target.

5. Characteristics of Drug Target
• The drug target is a biomolecule , normally a protein that
could exist in complex modality.
• The biomolecules have special sites that match other
molecule.
• Drug bind with the protein reversibly.
• Change in the biomolecule/protein structure follow the
physiological response.
• The physiological response play a major role in complex
regulation & have a therapeutic effect.
• Biomolecule’s activity, expression & structure might change
over duration of pathological process.
• These small molecule bind to the biomolecule are drugs.

6. Current Drug Targets

7. The most common drug targets of currently
marketed drugs include
 Protein
• G protein-coupled receptors (target of 50% of drugs)
• Enzymes (especially protein kinases, proteases, esterases,
and phosphatases)
• Ion channels
Ligand-gated ion channels
Voltage-gated ion channels
• Nuclear hormone receptors
• Structural proteins such as tubulin
• Membrane transport proteins
 Nucleic Acids

8. Receptor
 It is defined as a macromolecule or binding site located on the surface or inside
the effector cell that serves to recognize the signal molecule/ drug and initiate
the response to it, but itself has no other function.

9. A. G protein-coupled
receptors:
• A large family of membrane
receptor proteins with seven
transmembrane helical
segments, often associating
with G proteins to
transduce an extracellular
signal into a change in
cellular metabolism; also
called serpentine receptors
or heptahelical receptor .
Proteins as drug Target

10. Enzymes as receptor

11. Ion channels

12. Nuclear hormone receptors
 Nuclear hormone receptors are ligand-activated transcription factors that
regulate gene expression by interacting with specific DNA sequences
upstream of their target genes.
 Nuclear hormone receptor proteins form a class of ligand activated proteins
that, when bound to specific sequences of DNA serve as on-off switches for
transcription within the cell nucleus. These switches control the development
and differentiation of skin, bone and behavioral centers in the brain, as well
as the continual regulation of reproductive tissues.
 DNA Binding Domain (DBD)
 Ligand Binding Domain (LBD)

13. Membrane transport proteins
• A membrane transport protein (or
simply transporter) is a membrane
protein involved in the movement of ions,
small molecules, or macromolecules, such as
another protein, across a biological
membrane. Transport proteins
are integral transmembrane proteins; that is
they exist permanently within and span the
membrane across which they transport
substances. The proteins may assist in the
movement of substances by facilitated
diffusion or active transport. The two main
types of proteins involved in such transport
are broadly categorized as
either channels or carriers

14. Nucleic Acids
 The innate immune response
is critical for successful host
defence against virus
infection.
 Cell-intrinsic mechanisms
detect virus presence and
signal for the induction of
innate response genes such a
type I interferons (IFNs).
 Nucleic acids are often a
molecular signature of virus
infection and are recognised
by innate receptors including
toll-like receptors, RIG-I-like
receptors and cytosolic DNA
receptors.
 In addition to their protective
role in infectious disease, some
of these receptors have also
been implicated in
inflammatory conditions.

15. Home work
Find out the examples of different targets

16. Drug target identification

17. With the completion of the Human Genome Project, we now have the primary
amino acid sequence for all of the potential proteins in a typical human body.
However, knowledge of the primary sequence alone is not enough on which to
base a drug design project. For example, the primary sequence does not tell when
and where the protein is expressed, or how proteins act together to form a
metabolic pathway. Even more complex is the issue of how different metabolic
pathways are interconnected.
Ideally, the choice of which protein a drug will inhibit should be made based
on an analysis of the metabolic pathways associated with the disorder that the
drug is intended to treat. In reality, many metabolic pathways are only
partially understood. Furthermore, intellectual property concerns may drive
a company toward or away from certain targets.
Primary Sequence and Metabolic Pathway

18. 1. MetaCyc database available at http://metacyc.org.
2. KEGG Pathway Database available at
http://www.genome.ad.jp/kegg/pathway.html, which is interconnected
with the SEED Annotation/Analysis Tool at
http://theseed.uchicago.edu/FIG/index.cgi.
3. The ExPASy Biochemical Pathway
http://theseed.uchicago.edu/FIG/index.cgi is a front end to the digital
version of the Roche Applied Science “Biochemical Pathways” wall
chart.
Databases of metabolic pathways

19. Crystallography
Ideally, target protein structures should be determined by X-ray crystallography before
starting a drug design project. Additional information about protein conformational
changes can be obtained if structures both with and without a ligand soaked in the active
site are obtained. These are referred to as a “soaked” structure and an “apo” structure.
If both are not available, the soaked structure is preferred.
Unfortunately, proteins are notoriously difficult to crystallize, and membrane-bound
proteins are far more difficult than other proteins. However, the information gained from
crystallography is so valuable that extraordinary efforts and costs are justified. There are
even “structural genomics” projects that are attempting to crystallize all known proteins.

22. Drug Discovery Process
• Process starts with the identification of a molecular
target & then Target validation.
• The target validation confirms that interaction with the
target produce the desired change in the behaviour of
diseased cells.

23. Role of target identification
• A good target needs to be efficacious, safe, meet clinical and
commercial needs and above all, be ‘druggable’.
• When a biological molecule elicit a biological response which may
be measured both in vivo/vitro is known to be a druggable target .
• A good target identification increased confidence in the
relationship between target and disease.
Approaches to target identification
1. Direct biochemical method
2. Computational inference method
3. Genetic interaction method

24. Direct Biochemical Method
Direct methods involve labeling the protein or small
molecule of interest, incubation of the two populations
and direct detection of binding, usually following some
type of wash procedure.

25. Computational inference method
Comparsion of small molecule to the known reference
molecule is done.

26. Genetic interference method
⚫ Used to identify protein target.
⚫ Aim of this method is to compare two or more genomes in order to find
the similarities and differences
⚫ Hence identify potential drug target.
Genetic manipulation can also be used to identify protein targets by
modulating presumed targets in cells, thereby changing small-molecule
sensitivity.
Comparative genomics strategies aim to compare simultaneously two or
more genomes in order to identify similarities and differences, and hence
identify potential drug targets.

27. Tools for target identification and validation
1. Microarrays : Reliable technologies for addressing target identification and
validation are the foundation of successful drug development. Microarrays have been
well utilized in genomics/proteomics approaches for gene/protein expression profiling
and tissue/cell scale target validation.
2. Antisense technologies (including RNA interference technology) enable sequence-
based gene knockdown at the RNA level. Zinc finger proteins are a DNA
transcription targeting version of knockdown.
3. Chemical genomics and proteomics are emerging tools for generating phenotype
changes, thus leading to target and hit identifications. Target identification with
proteomics is performed by comparing the protein expression levels in normal and
diseased tissues.
4. NMR-based screening, as well as activity-based protein profiling, are trying to
meet the requirement of high-throughput target identification.

28. Microarrays
Microarray technology is a developing technology used to study the expression of
many genes at once. It involves placing thousands of gene sequences in known
locations on a glass slide called a gene chip. A sample containing DNA or RNA is
placed in contact with the gene chip
Target identification seeks to identify new targets, normally proteins (or DNA/RNA),
whose modulation might inhibit or reverse disease progression.
Current technologies enable researchers to attempt to correlate changes in gene
(genomics) and protein (proteomics) expression with human disease, in the hope of
finding new targets.
Assess gene and protein expression (via nucleic acid or protein microarrays) to
identify novel targets, and can also be used to validate the found targets at the tissue or
cell scale (via tissue or cell microarrays).

29. DNA Microarrays

30. Nucleic acid microarrays
Nucleic-acid microarrays, which primarily use short oligonucleotides (15–25 nt), long
oligonucleotides (50–120 nt) and PCR-amplified cDNAs (100–3000 base pairs) as array
elements, are overwhelmingly dominant because of the relatively easy synthesis and the
chemical robustness of DNA.
Data generated from genome sequencing projects in several organisms has provided the
opportunity to build comprehensive maps of transcriptional regulation. Array-based gene
expression analysis (immobilized DNA probes hybridizing to RNA or cDNA targets) has
enabled parallel monitoring of cellular transcription at the level of the genome.
Thus, nucleic-acid microarrays have had a significant impact on our understanding of
normal and abnormal cell biochemistry and, thus, on the choice of targets for drug design.
In oncology, data generated from high density oligonucleotide microarrays from
Affymetrix containing 62 907 probe sets have been analyzed and compared, to identify
97 genes as physiological targets of the retinoblastoma protein pathway, deregulation of
which is a hallmark of human cancer.
Further characterization of these genes should provide insights into how this pathway
controls proliferation, thus providing potential therapeutic targets.

31. Protein microarrays

32. Protein microarrays
Most drug targets are proteins, protein and peptide microarrays are set to have an
important impact on drug discovery. Protein arrays, an emerging yet very promising
technology, are now being used to examine enzyme–substrate, DNA–protein and
protein–protein interactions. By profiling the differential expression of proteins using
antibody arrays and correlating the changes to a disease phenotype, putative targets
(and biomarkers) to a particular disease can be identified, although to date, such
microarrays have not been used to their full potential because of difficulties with the
technology.
This strategy is based on the mechanism of the reaction between the ligands and
proteins, thus demonstrating the approach as an activity-based and high-throughput
method.
Further application of this method may lie on targeting known drugs or
biological active compounds.

33. Tissue and cell microarrays
An alternative to the use of whole-tissue specimens is the use of live cell microarrays,
which can be used to identify potential drug targets by functionally characterizing large
numbers of gene products in cell-based assays.
Antisense oligonucleotides
Complementary to a portion of a target mRNA molecule, oligonucleotides are the original
type of molecule used for blocking protein synthesis of the target mRNA, and thus
achieving the knockdown of the target gene.
One example is the identification of COX17 as a therapeutic target for non-small cell lung
cancer (NSCLC).
Chemical genomics and proteomics
Rather than finding drugs for targets in the conventional pharmaceutical approach, forward
chemical genomics, in a sense, finds targets for known drugs.
Its goal is to discover the specific molecular targets and pathways that are modulated by
particular chemical molecules (i.e. study the biochemistry underling the phenotype changes
induced by chemicals).

34. Thank you
Er. Rajan Rolta
Faculty of Applied Sciences and Biotechnology
Shoolini University,
Village Bhajol, Solan (H.P)
+91-7018792621 (Mob No.)
rajanrolta@shooliniuniversity.com