Drug Discovery:

Target Identification and

Validation
Florencio V. Arce, Jr., RPh, MSc, PhD, SRPharmS
Department of Pharmacy
Drug target
• Target – contains the molecular recognition site to which the drug will
bind.
• For the great majority of existing drugs the target is a protein
molecule, most commonly a receptor, an enzyme, a transport molecule
or an ion channel
• When the principle of drug discovery based on identified (then
pharmacological or biochemical) targets became established, the
pharmaceutical industry has recognized the importance of identifying new
targets as the key to successful innovation.
• It is estimated that the number of potential targets might be as high as
5000–10000.
• 120 drug targets accounted for the activities of compounds used
therapeutically, and estimated that 600–1500 ‘druggable’ targets exist
in the human genome.
Drug target
Drug target
• It is estimated that 100–150 high quality targets in the human
genome might remain to be discovered.
• The increased use of gene deletion mutant mice and si RNA for
gene knock down coupled with a bioinformatic approach to protein
characterization is starting to increase the reliability of target
identification.
• The largest groups of known drug targets are enzymes and G -
protein coupled receptors.
• Numerous subtype of transporters, receptor classes, and ion
channels
• Need for drugs that act on new targets arises because of the
major problem of drug resistance
Conventional strategies for finding new
drug targets
• Two main routes have been followed so far:
• Analysis of pathophysiology
• Analysis of mechanism of action of existing therapeutic drugs
• This biochemical (pathophysiological pathways) approach led to a
remarkable series of therapeutic breakthroughs in antibacterial,
anticancer and immunosuppressant drugs.
• Concept: First, understand the pathway leading from the primary
disturbance to the appearance of the disease phenotype, then
identify particular biochemical steps amenable to therapeutic inter-
vention, then select key molecules as targets.
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Conventional strategies for finding new
drug targets
• In some cases the target has emerged from pharmacological
rather than pathophysiological studies.
• The identification of drug targets by the ‘backwards’ approach –
involving analysis of the mechanism of action of empirically
discovered therapeutic agents – has produced some major
breakthroughs in the past.
• Its relevance is likely to decline as drug discovery becomes
more target focused, though natural product pharmacology will
probably continue to reveal novel drug targets.
Conventional strategies for finding new
drug targets
• ‘backward’ approach or
reverse pharmacology
• Drug target à HTS
à pre-clinical tests
New strategies for identifying drug
targets
• Drugs may intervene along the pathway from genotype to
phenotype, namely by altering gene expression, by altering the
functional activity of gene products, or by activating
compensatory mechanisms.
• Various genetic models (genomics-based approaches) have
been developed in different organisms for the identification of
new drug targets, and elucidating the mechanisms of action of
existing drugs.
one gene → one protein → one drug target
• Genome trawling will reveal more ‘single protein’ targets
 New strategies for identifying drug
targets
• Nearly all drug targets are proteins, and are, therefore, represented
in the proteome, and also as corresponding genes in the genome.

ü Proteome – complete
set of proteins
expressed by an
organism.

ü Genome – complete
set of genetic
information in an
organism.
Approaches towards identification of genes
that may encode novel drug targets
• ’Disease genes’, i.e. genes, mutations of which cause or predispose
to the development of human disease.
• ’Disease modifying’ genes. These comprise (a) genes whose altered
expression is thought to be involved in the development of the
disease state; and (b) genes that encode functional proteins, whose
activity is altered (even if their expression level is not) in the disease
state, and which play a part in inducing the disease state.
• ’Druggable genes’, i.e. genes encoding proteins likely to possess
binding domains that recognize drug like small molecules. Included
in this group are genes encoding targets for existing therapeutic and
experimental drugs.
Approaches towards identification of genes
that may encode novel drug targets
Disease genes
• The identification of genes in which mutations are associated
with particular diseases has a long history in medicine (e.g.
‘inborn errors of metabolism’ –phenylketonuria). There are 971
‘disease genes’.
• Much more common than single gene disorders are conditions
such as diabetes, hypertension, schizophrenia, bipolar
depressive illness and many cancers in which there is a clear
genetic component, but, together with environmental factors,
several different genes contribute as risk factors for the
appearance of the disease phenotype.
Disease-modifying genes
• These are nonmutated genes that are directly involved in the
pathophysiological pathway leading to the disease phenotype.
The phenotype may be associated with over or underexpression
of the genes, detectable by expression profiling, or by the over
or under activity of the gene product – for example, an enzyme
– independently of changes in its expression level.
• This is the most important category in relation to drug targets,
as therapeutic drug action generally occurs by changing the
activity of functional proteins, whether or not the disease alters
their expression level.
‘Druggable’ genes
• For a gene product to serve as a drug target, it must possess a
recognition site capable of binding small molecules.
• Binding domains in protein targets represent the current limit of the
druggable genome.
• To be useful as a starting point for drug discovery, a potential target
needs to combine ‘druggability’ with disease modifying properties.
• One new approach is to look at the characteristics of a typical human
protein drug target (e.g. hydrophobic, high length, signal motif
present) and to then look for these characteristics in a nontarget set
of proteins so as to identify new potential targets.
Target validation
• Target validation refers to the experimental approaches by
which a potential drug target can be tested and given further
credibility.
• It is an open-ended term, which can be taken to embrace
virtually the whole of biology, but for practical purposes the
main approaches are pharmacological and genetic.
• The ultimate test for the validity of a drug target is in the clinic,
where efficacy is or is not confirmed. Lack of clinical efficacy
causes the abandonment of roughly one third of drugs in Phase
II, reflecting the unreliability of the earlier surrogate evidence
for target validity.
Target validation: Pharmacological
approach
• The underlying question to be addressed is whether drugs that
influence the potential drug target actually produce the
expected effects on cells, tissues or whole animals.
• For example the metabotropic glutamate receptor (mGluR), was
identified as a potential target for a new indication (e.g. pain) its
validity could be tested by measuring the analgesic effect of
known mGluR antagonists in relevant animal models.
• A variant of the pharmacological approach is to use antibodies
raised against the putative target protein, rather than small -
molecule inhibitors.
Target validation: Genetic approach
• These approaches involve various techniques for suppressing the
expression of specific genes to determine whether they are critical to
the disease process.
• This can be done acutely in genetically normal cells or animals by the
use of antisense oligonucleotides or RNA interference, or con-
stitutively by generating transgenic animals in which the genes of
interest are either overactive or suppressed.
• Antisense oligonucleotides
• Antisense oligonucleotides bind to cellular mRNA and prevent its
translation. In principle this allows the expression of specific
genes to be inhibited, so that their role in the development of a
disease phenotype can be determined.
Target validation: Genetic approach
• RNA interference (RNAi)
• This technique depends on the fact that short lengths of
double stranded RNA (short interfering RNAs, or siRNAs)
activate a sequence specific RNA-induced silencing complex
(RISC), which destroys by cleavage the corresponding
functional mRNA within the cell.
• Thus specific mRNAs or whole gene families can be
inactivated by choosing appropriate siRNA sequences. Gene
silencing by this method is highly efficient in invertebrate
models, mammalian cells, and whole animals.
Target validation: Genetic approach
• Transgenic animals
• The gene knockout principle as a screening approach is used to
identify new targets.
• In principle, deletion or overexpression of a specific gene in vivo
can provide a direct test of whether or not it plays a role in the
sequence of events that gives rise to a disease phenotype.
• Requires a protocol of backcrossing the transgenic ‘founders’ into
the breeding strain has to proceed for several generations before
a genetically homogeneous transgenic colony is obtained (2
years).
• Cancer (p53 tumor suppressor gene); atherosclerosis (ApoE or
LDL-receptor genes; Alzheimer’s disease (amyloid precursor
protein or presenilin genes)