EARLY DRUG DISCOVERY

JACKLYN RISIA D. SAN GABRIEL, RPh, MSPharm

Drug discovery is a process which is intended to identify a small synthetic
molecule or a large biomolecule for comprehensive evaluation as a
potential drug candidate.

Doctortarget.com
identification of disease to be treated and its unmet medical need

Selection of a druggable molecular target and its validation

In vitro assay development followed by high throughput screening

of compound libraries against the target to identify hits

hit optimization to generate lead compounds that exhibit adequate

potency

selectivity towards the biological target in vitro and which demonstrate

efficacy in animal models of disease
From JP Hughes et al. Br J Pharmacol 2011
doi:10.1111/j.1476-5381.2010.01127.x/bph.1127
British Journal of Pharmacology © 2011 The British Pharmacological Society
the lead compounds are further optimized to improve
their efficacy and pharmacokinetics before they
advance towards drug development
Drug development process can be segregated into
preclinical and clinical development stages.
Preclinical Development
• Toxicological and safety pharmacology
• Establish the maximum safe concentrations in animals
• Determine the adverse effect potential of the drug-in-
development.
• Studies are conducted to finalize cost-effective
processes required for manufacturing the candidate
drug as well as deciding on its best formulation.
• If the candidate exhibits sufficient efficacy and safety
in preclinical evaluation – permission is sought from
drug regulatory agencies to initiate its clinical
development wherein the safety and efficacy of the
drug candidate is assessed in pilot and pivotal studies.
Innovative Drugs
• Discovery and development is Time and Cost intensive
• Currently approximately 12 years and an average of $1.8 billion is required to launch
a NEW DRUG.
• Decreasing trend in the number of innovative drugs obtaining Marketing Approval.
• Heightened scrutiny of the safety and efficacy of new drugs by regulatory agencies –
> leads to increased costs and prolonged development times.
• National Health services – adverse impact on their pricing
• Patent expirations and their generic substitutions have reduced the profits and
subsequent growth of the pharmaceutical industry
• Reduced investment in innovative research
• Adverse environmental impact of pharmaceuticals.

In spite of the fall in R&D productivity due to the above mentioned

concerns, there is still a high unmet need in the therapeutic areas of
cancer, Alzheimer’s disease, and diabetes, and there is a global
emergence of multidrug resistant bacterial infections for which
innovative drugs are urgently required.
 The trigger to initiate a drug discovery program is a medical condition whose
treatment is not satisfactorily addressed by currently available treatment modalities
 New drugs are needed either to treat a disease for which no
other treatment exists or which offer additional advantages over
existing treatments

Superior
Reduced
therapeutic
adverse effects
efficacy

Fewer drug-
Improved
drug
compliance
interactions

Overall
improvement in
the QOL of Px
 Small synthetic molecule
Biologically Active
Large molecular weight antibody
Compounds

Elicit their activity

Clinical effect by interacting with a

naturally existing molecular structure
TARGET
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. A good target needs to be
efficacious, safe, meet clinical and commercial needs
and, above all, be ‘druggable’.
Properties of a promising drug target
1. The target has a confirmed role in the pathophysiology of a
disease and/or is disease-modifying.
2.Target expression is not evenly distributed throughout the
body.
3. The target’s 3D-structure is available to assess druggability.
4. The target is easily ‘assayable’ enabling high-throughput
screening.
5. The target possesses a promising toxicity profile, potential
adverse effects can be predicted using phenotypic data.
6. The proposed target has a favorable intellectual property
(IP) status. (relevant for pharma companies)
The phenotypic approach to drug discovery falls within the realm of target
deconvolution, and involves exposing cells, isolated tissues, or animal models, to small
molecules to determine whether a specific candidate molecule exerts the desired effect
– which is observed by a change in phenotype.3 Whilst numerous animal models can be
used for the characterization of small molecules and small-scale drug screening
approaches, use of mammalian cells is often favored due to their compatibility with high-
throughput screening (HTS) and greater physiological relevance.

The phenotypic approach goes beyond individual proteins or nucleic acids and involves
the study of entire signaling pathways. The drug’s effect is determined before the
specific biological (drug) target that underlies the observed phenotypic response is
identified.
Phenotypic screening
• Compounds or antibodies are evaluated in cell-based
assays or animal models of disease
• Aim to identify compounds which elicit an anticipated
Change in the Phenotype
• Change in expression of a single or multiple proteins – In
vitro/In vivo
• Its molecular target is then determine by genetic
approaches
• Cloning techniques
• In silico approaches
• Chemical proteomic based approaches
• Affinity chromatography, Activity based protein profiling
& label free techniques.
Data Mining
• Available biomedical data has led to a significant increase in target
identification
• Bioinformatics approach
• Help in selecting and prioritizing potential disease targets
• Data – variety of sources
• Publications and patent information
• Gene expression data
• Transgenic phenotyping
• Compound profiling data

Identification approaches
• Examining mRNA/protein levels to determine whether they are expressed
in disease and if they are correlated with disease exacerbation or
progression
• Genetic associations – link between genetic polymorphisms and the risk of
disease or disease progression or is the polymorphism functional.
TARGET VALIDATION
•a potential drug target
needs to undergo the
process of validation
wherein its function in a
disease state is ascertained.
Antisense Technology
• Use of antisense technology is a popular route wherein short oligonucleotides (single stranded nucleic
acids), complimentary to a specific region of a messenger RNA of interest, are designed.
• The interaction of the oligonucleotides with the target mRNA results in disruption of translation and
subsequently impedes the synthesis of the protein.

RNA interference (RNAi) technology

• An alternative approach is RNA interference (RNAi) technology, wherein silencing of the target
• gene in a cell or an organism is triggered by introduction of a double stranded RNA (dsRNA) specific to
the gene
Genetically modified animals - phenotypic consequences of gene manipulation
• Employing genetically modified animals for target validation is an appealing methodology as it
• permits the scrutiny of the phenotypic consequences of gene manipulation. Development of knockouts,
• knock-ins, conditional knock-outs, and transgenic animals are instances of genetically modified animals.
• An animal lacking a particular gene from the embryonic stage is one approach to studying the in vivo
functions of diverse genes.
Transgenic animals
• Transgenic animals are also an attractive validation tool wherein a foreign gene is intentionally inserted in
their genome
Proteomics
• aims on the modulation of the activity of the target protein itself
Monoclonal antibodies
• are an excellent target validation tool as they interact with a larger region of the target
molecule surface, allowing for better discrimination between even closely related
targets and often providing higher affinity.
• However, antibodies cannot cross cell membranes restricting the target class mainly to
cell surface and secreted proteins.

Chemical genomics
• Chemical genomics can be defined as the study of genomic responses to chemical
compounds.
• The goal is the rapid identification of novel drugs and drug targets embracing multiple
early phase drug discovery technologies ranging from target identification and
validation, over compound design and chemical synthesis to biological testing.
• Chemical genomics brings together diversity-oriented chemical libraries and high-
information-content cellular assays, along with the informatics and mining tools
necessary for storing and analysing the data generated (reviewed in Zanders et al.,
2002).
• The ultimate goal of this approach is to provide chemical tools against every protein
encoded by the genome. The aim is to use these tools to evaluate cellular function prior
to full investment in the target and commitment to a screening campaign
From JP Hughes et al. Br J Pharmacol 2011
doi:10.1111/j.1476-5381.2010.01127.x/bph.1127
British Journal of Pharmacology © 2011 The British Pharmacological Society
From JP Hughes et al. Br J Pharmacol 2011 doi:10.1111/j.1476-5381.2010.01127.x/bph.1127
British Journal of Pharmacology © 2011 The British Pharmacological Society
HIT
• a compound which has the desired activity in a
compound screen and whose activity is confirmed
upon retesting. A variety of screening paradigms
exist to identify hit molecules
High throughput screening (HTS)
• screening of the entire compound library directly
against the drug target or in a more complex assay
system, such as a cell-based assay, whose activity
is dependent upon the target but which would
then also require secondary assays to confirm the
site of action of compounds (Fox et al., 2006).
High throughput screening (HTS)
• High throughput and other compound
screens are developed and run to
identify molecules that interact with the
drug target, chemistry programmes are
run to improve the potency, selectivity
and physiochemical properties of the
molecule, and data continue to be
developed to support the hypothesis
that intervention at the drug target will
have efficacy in the disease state.
HITS
• This screening paradigm involves the use of complex laboratory
automation but assumes no prior knowledge of the nature of
the chemotype likely to have activity at the target protein.

Focused or knowledge-based screening

• selecting from the chemical library smaller subsets of molecules
that are likely to have activity at the target protein based on
knowledge of the target protein and literature or patent
precedents for the chemical classes likely to have activity at the
drug target (Boppana et al., 2009).
• This type of knowledge has given rise, more recently, to early
discovery paradigms using pharmacophores and molecular
modelling to conduct virtual screens of compound databases
(McInnes, 2007).
Fragment screening
• generation of very small molecular weight
compound libraries which are screened at high
concentrations and is typically accompanied by
the generation of protein structures to enable
compound progression (Law et al., 2009).

Physiological screening
• This is a tissue-based approach and looks for a
response more aligned with the final desired in
vivo effect as opposed to targeting one specific
molecular component.
 Pharmacological relevance
Assay Development
Reproducibility
Factors

Assay costs

Assay Quality

Effects of compounds in the assay

CONFIRMATION OF HITS
• The identified hits are subjected to confirmatory evaluation using the same assay conditions which were
employed during Hit Identification.
• Ascertain that the activity is linked to the anticipated mechanism and is not due to artifacts.
• Detecting false positives = counter-screening assay
False Positives
• Hits are evaluated for their activity against an alternative member of the target family under identical
assay conditions and if the hit demonstrates similar activity then it is most likely a false positive.
• precipitation of the small molecule as well as aggregate formation
identifying compounds that may produce activity based on detrimental mechanisms
• Toxicity
• Establishing dose-response relationships is also essential as an all-or-none response may point to non-
specific effects or interaction with any other constituent of the assay condition.
• estimation of half maximal inhibitory concentration (IC50)
• half maximal effective concentration (EC50)
• Experiments examining the nature of binding of the actives at this stage are also advantageous as they
filter out compounds showing non-competitive interaction with the target and which will not be the
preferred candidates for evaluation in a clinical setting.

ASSAY
• the hits may be screened in a secondary cell-based or a functional assay in which the target has been
proven to play a role in order to ascertain their efficacy.
Structure-activity relationship (SAR)
• studies the association between the three-dimensional
structure of a molecule with its biological, pharmacological,
and chemical activity on the receptors.
• Often, the new parent molecules developed to treat
particular disease express low affinity, less specificity, and
high toxicity.
• improve affinity and specificity, knowledge about the SAR of
the parent molecule is important.
• Majority of drugs with better efficacy and safety used
clinically are being developed by meticulous analysis of SAR
of the parent molecule and modification of its structure.
Implications of Structure-Activity Relationship
• Determination of chemical group (pharmacophore) responsible for affinity
of the molecule to its receptor. By altering the structure of
pharmacophore, the affinity of the molecule to the desired receptor can
be increased (achieving high affinity resulting in better potency).
• Modification of chemical group responsible for interactions with
unwanted receptors thereby improving the specificity of drug (lesser
incidences of side effects).
• Slight alteration in chemical structure of parent molecule can improve the
pharmacokinetic properties of the drug which would benefit by ease of
administration, long duration of action, better tolerability for patients in
hepatic and renal co-morbid conditions, and others.
• Several antagonists of particular receptors were developed by minor
alterations in the natural agonist after the detailed SAR analysis of natural
ligands.
• With the aid of SAR analysis, development of agonists with improved
pharmacokinetic and pharmacodynamics profile is possible with
modifications in their structure.
HIT-TO-LEAD PHASE
•LEAD GENERATION
•refine each hit series to try to produce
more potent and selective compounds
which possess PK properties adequate
to examine their efficacy in any in vivo
models that are available.
 The Rule of 5 – Two decades later

It’s now over 20 years since Pfizer’s Chris Lipinski introduced the Rule of 5 to
describe drug-like molecules. Rather than a set of hard and fast rules, it provides
a guideline that points designers towards molecules whose properties make them
more likely to succeed in the clinic.

To be drug-like, a molecule should have no more than five

hydrogen bond donors, no more than 10 hydrogen bond
acceptors, a molecular weight less than 500 Da, and log P
below 5 to ensure it is not too lipophilic.
The goal of lead optimization is to generate preclinical development candidates by
improving the shortcomings of the lead structure by chemical modifications.
Generally, the aim is to enhance the physicochemical and ADME properties and
minimize the toxicity liabilities so that a potentially safe compound with favorable
pharmacokinetics is identified.
LEAD OPTIMIZATION
• The object of this final drug discovery phase is to maintain favourable
properties in lead compounds while improving on deficiencies in the lead
structure.
• Compounds at this stage may be deemed to have met the initial goals of
the lead optimization phase and are ready for final characterization before
being declared as preclinical candidates.
• Discovery work does not cease at this stage. The team has to continue to
explore synthetically in order to produce potential back up molecules, in
case the compound undergoing further preclinical or clinical
characterization fails and, more strategically, to look for follow-up series.
• Molecules need to be examined in models of genotoxicity such as the
Ames test and in in vivo models of general behaviour such as the Irwin’s
test.
• High-dose pharmacology, PK/PD studies, dose linearity and repeat dosing
PK looking for drug-induced metabolism and metabolic profiling all need
to be carried out by the end of this stage.
• chemical stability issues and salt selection for the putative drug substance.
10% of small molecule projects within industry might make the transition to candidate,
failing at multiple stages.
These can include the
(i) inability to configure a reliable
assay;
(ii) no developable hits obtained from the HTS;
(iii)compounds do not behave as desired in secondary or native
tissue assays;
(iv) compounds are toxic in vitro or in vivo;
(v) compounds have undesirable side effects which cannot be
easily screened out or separated from the mode of action of
the target;
(vi) inability to obtain a good PK or PD profile in line with the dosing regime required in
man, for example, if require a once a day tablet then need the compound to have a
half-life in vivo suitable to achieve this;
(vii) inability to cross the blood brain barrier for compounds whose target lies within
the central nervous system.
The attrition rate for protein therapeutics, once the target has been identified, is much
lower due to less off target selectivity and prior experience of PK of some proteins, for
example, antibodies.
 CRITERIA TO SELECT A CLINICAL CANDIDATE
Chemical properties It should be a stable molecule whose synthesis is simple and can be
scaled up with ease.
Physicochemical It should observe Lipinski rule of 5 and should have acceptable
properties solubility.
Pharmacological It should bind with the target site with high affinity, should
properties: demonstrate selectivity for its molecular target and should elicit potent
functional effect
in vitro. Efficacy of the compound should be demonstrable in animal
model of human disease.
Pharmacokinetic It should possess acceptable bioavailability, adequate half-life and
properties: proper distribution in animals. Metabolic pathways of the compound
should be well characterized and activities of metabolites should be
evaluated.
Safety and toxicity It should be devoid of cardiac toxicity (hERG binding), genotoxicity, and
potential: hepatotoxicity, and should demonstrate an acceptable profile for
induction and inhibition of cytochrome P450 enzymes. Ultimately it
should be devoid of any serious animal toxicity.
 CONCLUSION
Pharmaceutical industry is currently under immense pressure
due to rising costs, pricing, and risks associated with drug
discovery and development. However, due to high unmet
medical needs, the disease treatment will continue to be
determined by innovation generated by the industry in
collaboration with academic institutions and other modes of
public-private partnerships. Intellectual property protection,
however, is important in supporting pharmaceutical R&D. New
developments in science and technology and other innovative
and emerging approaches to improve R&D productivity need to
be adopted with a long-term approach in order to be truly
supportive of novel drug research.
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