Assignment on:

“Target identification in drug discovery”

Submited to: Submited by:

Dr. Durg Vijay Singh S. M. Shayez Karim

Central University of South Bihar Saima Firdaus

Patna- 800014 (BIHAR), INDIA Shweta Kumari

Swati Kumari

Introduction:

“Identifying the biological origin of a disease, and the potential targets for

intervention, is the first step in the discovery of a medicine.”

A target is a broad term which can be applied to a range of biological entities which may

include for example proteins, genes and RNA.

Target identification is the process of identifying the direct molecular target – for

example protein or nucleic acid – of a small molecule. In clinical pharmacology, target

identification is aimed at finding the efficacy target of a drug/pharmaceutical or other

xenobiotic.

There is need to find a protein (e.g. receptor) or gene associated with a disease with

which a potential drug interacts – the so-called targets.

Note: Not every target is equally capable of affecting the course of a disease.

Source: http://image.slidesharecdn.com/introduction-to-drug-discovery-1197991446435040-4/95/introduction-to-drug-discovery-4-728.jpg?cb=1197962647

The drug discovery process starts with the identification of a molecular target and the

next is the Target Validation. During target validation, its association with a specific

disease and its ability to regulate biological processes is tested in the body. The target

validation confirms that interactions with the target produce the desired change in the

behavior of diseased cells.

It is the critical step in Drug discovery process. Identification of new drug targets, target

validation, biochemical assay development followed by LEAD identification provides

very important input in the development of new potential drug candidate.

Figure: steps of drug discovery

Characteristics of Drug Target

1. A specific drug target might have the following characteristics:

2. The drug target is a biomolecule(s), normally a protein that could exist in isolated or

complex modality.

3. The biomolecules have special sites that match other molecules (commonly small

molecules with special structures). These molecules could be endogenous or extraneous

substances such as chemical molecules (drugs).

4. The bio-molecular structure might change when the biomolecule binds to small

molecules and the changes in structure normally are reversible.

5. Following the change in the biomolecule’s structure various physiological responses

occur and induce regulation of the cell, organ, tissue, or body status.

6. The physiological responses triggered by the changes in biomolecule structure play a

major role in complex regulation and have a therapeutic effect on pathological

conditions.

7. The expression, activity, and structure of the biomolecule might change over the duration

of the pathological process.

8. Small molecules binding to the biomolecules are drugs.

Traditional Drug Discovery v/s New Strategies in Drug discovery

The path to identifying and validating the drug candidate molecule is not a one size- fits-

all-diseases strategy.

The first stage in drug discovery process is to understand the disease mechanism, using

cellular and genetic approaches, in order to identify potential drug targets.

Role of target identification in drug discovery:

Target identification and mechanism of action studies play an important role in small-

molecule discovery.

A good target needs to be efficacious, safe, meet clinical and commercial needs and,

above all, be ‘druggable’. A ‘druggable’ target is accessible to the putative drug

molecule, be that a small molecule or larger biological and upon binding, elicit a

biological response which may be measured both in vitro and in vivo.

Good target identification and validation enables increased confidence in the relationship

between target and disease and allows us to explore whether target modulation will lead

to mechanism-based side effects.

Current therapy is based upon less than 500 molecular targets of about 10000 possible

targets:

a. 45% of which are G-protein coupled receptors

b. 28% are enzymes

c. 11% are hormones and factors

d. 5% are ion channels

e. 2% are nuclear receptors

Figure: Current Drug Targets - few target classes; based on 483 drugs in Goodman and Gilman's "The Pharmacological

basis of therapeutics"

Source: Jürgen Drews, et al.Drug Discovery: A Historical Perspective. Science 287, 1960 (2000);DOI:

10.1126/science.287.5460.1960

Target identification can be approached by direct biochemical methods, genetic

interactions, or computational inference. Combinations these approaches may be required

to fully characterize mechanisms of small-molecule action.

Accordingly, the Broad Institute uses a multi-faceted approach to the target identification

problem in the context of genome-based drug discovery, including:

a. Quantitative proteomics based on mass spectrometry

b. Genetic complementation of small-molecule effects using RNA interference

c. Computational inference by connectivity analysis using reference compounds

A forward genetics (or classical genetics) approach is characterized by identifying, often

under experimental selection pressure, a phenotype of interest, followed by identification

of the gene (or genes) responsible for the phenotype.

Modern molecular biological methods, particularly genetic engineering approaches, have

given rise to reverse genetics (sometimes equated with molecular genetics), in which a

specific gene of interest is targeted for mutation, deletion or functional ablation (for

example, with RNAi11), followed by a broad search for the resulting phenotype.

Enzymes

28%

Nuclear

Receptors

2%

DNA

2%

Unknown

7%

Ion-

Channels

5%

Hormones &

factors

11%

Receptors

(GPCRs)

45%

Figure. Mechanism-of-action and target identification in chemical genetics. (a) Target-based approaches

(reverse chemical genetics) begin with target validation, in which a role is established for a protein in a

pathway or disease, followed by a biochemical assay to find candidate small molecules; mechanism-of-

action studies are still required to validate cellular activities of candidates and evaluate possible side

effects.(b) Phenotype-based approaches (forward chemical genetics) begin with a phenotype in a model

system and an assay for small molecules that can perturb this phenotype; candidate small molecules must

then undergo target-identification and mechanism-of-action studies to determine the protein responsible for

phenotypic change.

Drugs Target at a molecular level:

The main molecular targets for drugs are proteins (mainly enzymes, receptors, and

transport proteins) and nucleic acids (DNA and RNA).

These are large molecules (macromolecules) that have molecular weights measured in the

order of several thousand atomic mass units. They are much bigger than the typical drug,

which has a molecular weight in the order of a few hundred atomic mass units. The

interaction of a drug with a macromolecular target involves a process known as binding.

There is usually a specific area of the macromolecule where this takes place, known as

the binding site.

Typically, this takes the form of a hollow or canyon on the surface of the macromolecule

allowing the drug to sink into the body of the larger molecule. Some drugs react with the

binding site and become permanently attached via a covalent bond.

However, most drugs interact through weaker forms of interaction known as

intermolecular bonds .These include:

o Electrostatic or ionic bonds,

o Hydrogen bonds,

o Van der Waals interactions,

o Dipole–dipole interactions, and

o Hydrophobic interactions.

(It is also possible for these interactions to take place within a molecule, in which case they are

called intra molecular bonds.)

Note: None of these bonds is as strong as the covalent bonds that makeup the skeleton of a

molecule, and so they can be formed and then broken again. This means that, equilibrium

takes place between the drug being bound and unbound to its target.

The binding forces are strong enough to hold the drug for a certain period of time to let it

have an effect on the target, but weak enough to allow the drug to depart once it has done

its job. The length of time the drug remains at its target will then depend on the number

of intermolecular bonds involved in holding it there.

Drugs that have a large number of interactions are likely to remain bound longer than

those that have only a few. The relative strength of the different intermolecular binding

forces is also an important factor. Functional groups present in the drug can be important

in forming inter-molecular bonds with the target binding site. If they do so, they are

called binding groups.

However, the carbon skeleton of the drug also plays an important role in binding the drug

to its target through van der Waals interactions. As far as the target binding site is

concerned, it too contains functional groups and carbon skeletons which can form

intermolecular bonds with ‘visiting’ drugs. The specific regions where this takes place

are known as binding regions.

The study of how drugs interact with their targets through binding interactions and

produce a pharmacological effect is known as pharmacodynamics.

Approaches to target identification:

There are three distinct and complementary approaches for discovering the protein target of a

small molecule:

1. Direct biochemical methods -

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.

2. Genetic interaction methods –

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.

3. Computational inference methods –

Target hypotheses, in contrast, can be generated by computational inference,

using pattern recognition to compare small-molecule effects to those of known reference

molecules or genetic perturbations.

Tools for target identification and validation:

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.

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.

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.

NMR-based screening, as well as activity-based protein profiling, are trying to meet the

requirement of high-throughput target identification.

Microarrays:

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).

Nucleic acid microarrays

Today, 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.

Protein microarrays

Because 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.

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.

Figure: Tools for target identification and validation.

Antisense technology:

A key strategy in target validation is to determine what happens, with respect to phenotype

and/or the expression of other genes in cells or model organisms, if a gene of interest is either

deleted or its activity is inhibited.

Gene knockout mimics the activity of a drug that completely inhibits the normal function of the

gene’s product.

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).

RNA interference

RNA interference (RNAi) is another type of technology involving sequence-specific RNA for

use in gene knockdown.

This approach avoided some limits such as selectivity of mutational targeting, complexities of

anatomically based phenotypic analysis, or difficulties in subsequent gene identification, and

should make possible the systematic identification of components within each pathway, thus

leading to potential therapeutic targets.

Zinc finger proteins

Zinc finger proteins (ZFPs) have remarkable versatility for recognizing different sequences of

DNA, and variations in the amino acid sequence of the C2H2 domains allow them to be targeted

to different locations in the genome.

Each zinc finger is a short stretch of 30 amino acids, containing two conserved cysteines and two

conserved histidines. These proteins have been used as the DNA-binding domains of novel

transcription factors (ZFP TFs).

ZFP TFs can be applied to potential new drug target validation in two ways.

a. One direct way involves designing ZFPs to validate the phenotypic effects of activating

or repressing a gene.

b. Alternatively, libraries of ZFP TFs might be used to screen cells for desired phenotypic

changes.

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).

Tagged library approach

Various kind of chemicals have been used to generate novel chemotypes and once an effect is

found, the next step is to identify the biological target using an affinity matrix made of the

immobilized hit compound.

High-throughput NMR-based screening is also utilized for fast identification of drug–target

interaction.

Affinity Chromatography as a Classical Method for Target Identification

Despite the large number of target identification techniques described to date, affinity

chromatography remains the most widely used method. The typical project begins with

structure–activity relationship (SAR) studies in which various functional groups of the small

molecule of interest are modified or removed to determine which one(s) are dispensable for drug

activity.

The primary limitation of affinity chromatography is the need to derivatize the small molecules

of interest. SAR studies are time-consuming and require extensive medicinal chemistry expertise

that is often lacking in the academic laboratories performing forward chemical genetics and

phenotypic small molecule screens.

A recent major advance in the affinity chromatography approach for target identification takes

advantage of the quantitative capability of SILAC to drastically increase the sensitivity of this

approach for target identification.

Drug affinity responsive target stability (DARTS)

DARTS is the method for target identification that relies on drug-induced protease resistance.

Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward

approach to identify potential protein targets for small molecules. It relies on the protection

against proteolysis conferred on the target protein by interaction with a small molecule.

The basis for DARTS is that a protein becomes stabilized upon binding to a small molecule

compound or other ligand, which leads to decreased susceptibility of the target protein to

degradation by proteases. This decreased proteolysis is specific to the target protein(s) and

occurs for both high and low affinity compounds. Moreover, DARTS works especially well

using extremely complex protein samples such as whole cell lysates where non-specific protein-

ligand interactions are minimized due to the large number and variety of proteins in the mixture.

DARTS is advantageous because any small molecule can be used in its native form, meaning no

Structure-Activity Relationship (SAR) studies or chemical modifications to the ligand are

necessary for target identification.

DART- Overview:

First, the source of protein must be chosen. Generally any cell type that is sensitive to the

biological effects of the small molecule can be used.

Second, any small molecule believed to bind proteins should be suitable for DARTS (including

drug-like small molecules that are not susceptible to proteolysis, and peptide ligands that are

themselves susceptible to proteolysis but whose binding to target proteins would protect them

from proteolysis), but the concentration range of small molecule to use is also an important

consideration. Given that the targets of most small molecules being used for DARTS are

unknown, the binding affinities will also not be known. Therefore, one can only estimate the

binding affinity to the most relevant target(s) based upon the EC50 of the compound, although

this correlation may not be valid for all compounds and biological systems. Since the EC50 gives

only a rough estimate of binding affinity, it is done initially using a concentration of the

compound that is 10-fold higher than the EC50. Using a concentration of compound that is

significantly higher than the KD will help ensure maximal protection of the target protein from

proteolysis by saturating the protein with ligand.

It is particularly useful for the initial identification of the protein targets of small molecules, but

can also be used to validate potential protein-ligand interactions predicted or identified by other

means and to estimate the affinity of interactions.

DART v/s Affinity Chromatography:

DARTS offers an unprecedented ability to identify new proteins targeted by small molecules. It

is similar to affinity chromatography in that both are affinity based methods that start with

complex protein samples and selectively enrich the target protein(s) while depleting all non-

target proteins.

However, whereas affinity chromatography utilizes positive enrichment by selectively pulling

out the target proteins and leaving behind non-targets, DARTS uses negative enrichment by

digesting away non-target proteins while leaving behind the target proteins that are rendered

protease-resistant.

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