What is a “hit” in drug discovery?

 In drug discovery, a "hit" refers to a chemical compound that exhibits a detectable

biological activity against a specific target molecule or a biological assay.

 These compounds are considered promising starting points because they show initial
interactions with the target of interest, indicating the potential to be developed into
more potent and selective drug candidates through further optimization.
A hit could be defined as a compound that meets preconceived threshold criteria for 'activity' in the primary
screening assay. Materials that are screened include natural products, synthetic compounds from any source, and
custom-made combinatorial 'libraries.' The choice of materials for screening can be random, but may be limited
or targeted on the basis of theory or experience. Very few hit compounds progress further to become leads (see
next section), but any structure-activity data might be useful in defining the structural requirements for activity.
A lead is defined as a compound that is reproducibly a 'hit', whose structure is deemed to be modifiable into analogues
with 'drug-like' physicochemical properties. These materials will meet the target (rather than threshold) criteria of the
primary biological assay, but may still not be suitable for pharmaceutical use — they may be weakly active, unstable, too
expensive to produce, have off-target effects or be unpatentable, but they can still guide chemists to a final drug structure.
The following criteria are established in identifying lead compounds:
potency: A hit compound is initially re-tested under the original assay conditions. Then its potency is quantified
using a dose response curve, generated by measuring the effect of the compound at different concentrations. The
potency is quantified in the IC50 or EC50 value.
•Efficacy: Activity will be measured in other assays. At least one of these will be cell-based, to confirm that the
compound can pass through biological membranes on its way to the target.

•Tractability: Medicinal chemists will assess whether the hit structure is amenable to chemical modification, i.e. that
analogues with more drug-like properties will be accessible.

•Availability: A hit compound is assessed for ease of synthesis at a reasonable cost, bearing in mind the eventual
requirement for large-scale production.

•Patentability: The intellectual property (IP) rights are verified using appropriate databases.
1. Endogenous Ligands-
i.e.- Substrate for enzyme and transportersor agonist for receptors

Norepinephrine
2. Natural Products-

(neurotransmitter) HMG- CoA( metabolite)

3. Animals

 Animal cartilage
 Gelatin capsules or gummy vitamins chondroitin
 Animal bones
 Calcium
 Animal blood Structure of Fe-
porphyrin subunit of
 Heme iron heme B.
4. Microorganisms

Penicillin
 Screening strategies to identify hit molecules

 High throughput
 Focused screen
 Fragment screen
 Virtual screen
 NMR screen
 Physiological screen
High throughput screening (HTS) involves the 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).
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 involves 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

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

Fragment screening involves the 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

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.
 Assay development

cell-based assays have been applied to target classes such as membrane receptors, ion channels and nuclear
receptors and typically generate a functional read-out as a consequence of compound activity

biochemical assays, which have been applied to both receptor and enzyme targets, often simply measure the
affinity of the test compound for the target protein

Both assay paradigms have been used successfully to identify hit and candidate molecules

Multiple assay formats have been enabled to support compound screening.

The choice of assay format is dependent upon the biology of the drug target protein, the equipment infrastructure
in the host laboratory, the experience of the scientists in that laboratory, whether an inhibitor or activator
molecule is sought and the scale of the compound screen

For example compound screening assays at GPCRs have been configured to measure the binding affinity of a radio-
or fluorescently labelled ligand to the receptor, to measure guanine nucleotide exchange at the level of the G-
protein, to measure compound-mediated changes in one of a number of second messenger metabolites including
calcium, cAMP or inositiol phosphates or to measure the activation of downstream reporter genes
 Factors important in Assay Development
Pharmacological relevance of the assay. If available, studies should be performed using known ligands with
activity at the target under study, to determine if the assay pharmacology is predictive of the disease state and
to show that the assay is capable of identifying compounds with the desired potency and mechanism of action

Reproducibility of the assay. Within a compound screening environment it is a requirement that the assay is
reproducible across assay plates, across screen days and, within a programme that may run for several years,
across the duration of the entire drug discovery programme.

Assay costs. assay reagents and assay volumes are selected to minimize the costs of the assay.

Assay quality. Assay quality is typically determined according to the Z’ factor (Zhang et al., 1999). This is a
statistical parameter that in addition to considering the signal window in the assay also considers the variance
around both the high and low signals in the assay. The Z factor has become the industry standard means of
measuring assay quality on a plate bases. The Z factor has a range of 0 to 1
an assay with a Z factor of greater than 0.4 is considered appropriately robust for compound screening
although many groups prefer to work with assays with a Z factor of greater than 0.6.

Effects of compounds in the assay. Chemical libraries are typically stored in organic solvents such as ethanol or
dimethyl sulphoxide (DMSO). Thus, assays need to be configured that are not sensitive to the concentrations of
solvents used in the assay. Typically, cell-based assays are intolerant to solvent concentrations of greater than
1% DMSO whereas biochemical assays can be performed in solvent concentrations of up to 10% DMSO. Studies
are also performed to establish the false negative and false positive hit rates in the assay
When screened to detect agonist ligands the hit rates in the aequorin assay are typically less than 0.5% of compounds
screened with a statistical assay cut-off of 5% or less of the agonist signal seen with a standard agonist ligand. In this assay
format false positive and false negative hit rates are very low. For antagonist screening the hit rate in the aequorin assay is
typically of 2–3% of compounds screened with an activity cut-off of greater than 25% inhibition. This is a common
phenomenon of all screening assays. Hit rates in antagonist or inhibitor format tend to be higher than hit rates in agonist
assays as antagonist assays, which are defined by detection of a decrease in assay signal, will also detect compounds that
interfere in signal generation.

In the agonist assay (left panel), no drug response is represented in red, the response to a maximal concentration of
the ligand histamine in blue and compound data in yellow. As is typically seen in agonist assays, the hit rate is very
low due to the absence of false positives. In the antagonist assay (right panel), the response to histamine in the
absence of test compound is represented in red (basal response), the response to a maximal concentration of a
histamine antagonist in blue (100% inhibition) and compound data in yellow. As is typically seen in a cell-based
inhibitor assay, there is significant spread of the compound data due to a combination of assay interference and
compound activity. True actives correlate in the range 40% to 100% inhibition. Both assays have excellent
Quality control (QC) in high throughput screening. To ensure the control of screening data in compound screening campaigns
each assay plate typically contains a number of pharmacological control compounds. (A) Each 384-well plate contains 16
wells containing a low control and a further 16 wells containing an EC100 concentration of a pharmacological standard which
are used to calculate the Z’ factor (reference Zhang et al., 1999). Plates that generate a Z’ factor below 0.4 are rescreened.
(B) Each plate also contains 16 wells of an EC50 concentration of a pharmacological standard to monitor the variance in the
assay (diamonds). (C) A heat map is generated for all plates that pass the pharmacological standard QC to monitor the
distribution of activity across the assay plate. One would expect to see a random distribution of activity across the screening
plate. A plate such as the one presented would be failed and rescreened due to the active wells clustering in the centre of
the plate.
Hit confirmation
• After hits are identified from a high throughput screen, the hits are confirmed and evaluated using the following
methods:
Confirmatory testing: compounds that were found active against the selected target are re-tested using the same
assay conditions used during the HTS to make sure that the activity is reproducible.
• Dose response curve: the compound is tested over a range of concentrations to determine the concentration
that results in half maximal binding or activity (IC50 or EC50 value respectively).
Orthogonal testing: confirmed hits are assayed using a different assay which is usually closer to the target
physiological condition or using a different technology.
• Secondary screening: confirmed hits are tested in a functional cellular assay to determine efficacy
Synthetic tractability: medicinal chemists evaluate compounds according to their synthesis feasibility and
other parameters such as up-scaling or cost of goods.
Biophysical testing: nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), dynamic light
scattering (DLS), surface plasmon resonance (SPR), dual polarisation interferometry (DPI), microscale
thermophoresis (MST) are commonly used to assess whether the compound binds effectively to the target,
the kinetics,thermodynamics, and stoichiometry of binding, any associated conformational change and to rule
out promiscuous binding.
Hit ranking and clustering: Confirmed hit compounds are then ranked according to the various hit
confirmation experiments.
Freedom to operate evaluation: hit structures are checked in specialized databases to determine if they are
patentable
 Defining a hit series

Once a number of hits have been obtained from virtual screening or HTS, the first role for the drug discovery team is to try
to define which compounds are the best to work on.
from a large library, a team will likely be left with many possible hits which they will need to reduce, confirm and cluster
into series
There are several steps to achieving this
First, compounds that are known by the library curators to be to be frequent hitters in HTS campaigns need to be removed
from further consideration.
Second, a number of computational chemistry algorithms have been developed to group hits based on structural similarity

these algorithms allows the selection of hits for progression based on chemical cluster, potency and factors such as ligand
efficiency which gives an idea of how well a compound binds for its size
The next phase is to generate dose–response curves in the primary assay for each hit, preferably with a fresh sample of the
compound.
Compounds which give an all or nothing response are not acting in a reversible manner and indeed may not be binding to
the target protein at all, high concentrations arising from an interaction between the sample and another component of
the assay system.

Obtaining a dose–response curve allows the generation of a half maximal inhibitory concentration which is used to
compare of the potencies of candidate compounds.
With reliable dose–response curves generated in the primary assay for the target, the stage is set to examine the surviving
hits in a secondary assay such as second messenger assay or in a tissue-or cell-based bioassay
Throughout the confirmation process, medicinal chemists would be looking to cluster compounds into groups which could
form the basis of lead serie
consideration will be given to the properties of each cluster such as whether there is an identifiable structure–activity
relationship (SAR)

representative examples of each of these mini-series will be subjected to various in vitro assays designed to provide
important information with regard to absorption, distribution, metabolism and excretion (ADME) properties as well as
physicochemical and pharmacokinetic (PK) measurements (see T
 Hit Expansion

Hit expansion Following hit confirmation, several compound clusters will be chosen according to their
characteristics in the previously defined tests. An Ideal compound cluster will contain members that
possess: high affinity towards the target (less than 1 µM) selectivity versus other targets significant
efficacy in a cellular assay druglikeness (moderate molecular weight and lipophilicity usually estimated
as ClogP). Affinity, molecular weight and lipophilicity can be linked in single parameter such asligand
efficiency and lipophilic efficiency. low to moderate binding to human serum albumin low interference
with P450 enzymes and P- glycoproteins low cytotoxicity
metabolic stability
high cell membrane permeability
high water solubility (above 10 µM) chemical stability synthetic tractability patentability The project team will usually
select between three and six compound series to be further explored.
The next step will allow the testing of analogous compounds to determine aquantitative structure-activity relationship
(QSAR). Analogs can be quickly selected from an internal library or purchased from commercially available sources
("SAR by catalog").
Medicinal chemists will also start synthesizing related compounds using different methods such as combinatorial
chemistry, high-throughput chemistry, or more classical organic chemistry synthesis.
 Hit-to-lead phase

The aim of this stage of the work is to 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 work now consists of
intensive SAR investigations around each core compound structure, with measurements being made to establish the
magnitude of activity and selectivity of each compound
structure-based drug design techniques using molecular modelling and methodologies such as X-ray crystallography
and NMR can be applied to develop the SAR faster and in a more focused way

Solubility and permeability assessments are crucial in ruling in or out the potential of a compound to be a drug, that
is, drug substance often needs access to a patient’s circulation and therefore may be injected or more generally has to
be adsorbed in the digestive system. Deficiency in one or other parameter in a molecule can sometimes be put right.
A compound that lacks both these properties is very unlikely to become a drug no matter how potent it is in the
primary screening assay.
Microsomal stability is a useful measure of the ability of in vivo metabolizing enzymes to modify and then remove a
compound.
CYP450 inhibition is examined as, among other things, it is an important predictor of whether a new compound might
have an influence on the metabolism of an existing drug with which it may be co-administered.
 High throughput Screening
 Screening of the entire compound library directly against the drug target

 Robotic systems and automated assays.

 Rapidly screen a large number of compounds

 Compounds that exhibit promising activity are referred to as "hits."

High-throughput screening robots

 Assay Development

 Assays are investigative procedures that qualitatively assess a compound or examine a compound's
effects on identified molecular, cellular, or biochemical targets.

 In the following stage of drug development, biological assays and compound screening assays are
created. These assays are used to identify compounds that have a desired activity at the drug target.
These compounds are referred to as "hit" molecules.

 During the initial phase of hit compound identification, termed high throughput screening (HTS), a
compound library that contains many potential hit molecules is tested to identify any compounds
with the desired activity towards the target.
Cont.

 Further assays are required to retest the hit molecule's activity at the target.

 Finally, cell-based assays are used to examine a drug's toxicity, safety profile, and efficacy.

 Every drug that is developed undergoes a unique series of assays that are specifically designed
and organized for the drug target and compound in question. This process is termed assay
development.
Factors important in Assay Development
FACTOR
IMPORTANCE IN ASSAY DEVELOPMENT
Relevance
Research should be conducted to examine the ability of the assay to: 1.Predict the specific disease
state. 2.Identify compounds that exhibit an appropriate mechanism of action and strength.
Reproducibility
In a compound screening environment, an assay must be reproducible. This means that feasible
reproducibility exists across assay plates, screen days and the full duration of the specific drug
discovery program.
Quality
Z'-factor is calculated to measure the power or quality of an HTS assay. The signal window and
variance of negative and positive signals is used in this calculation.
The Z-factor must be >0.4 to be considered acceptable for use, with some researchers preferring a
Z'-factor of >0.6.
Interference
Assays must be designed that consider the effects of compounds found in the assay, such as the
solvents used.
 High Throughput Screening (HTS)
 HTS is a process to accelerate drug discovery, which involves a brute force approach where tens of
thousands of compounds (Compound libraries) are tested against a particular target daily.

 This method is very valuable to early drug discovery.

 Compounds are being tested using a quantitative bioassay via the use of automation, miniaturized
assays, micro fluidic chips, sub nano litre dispensing, fluorescence, large-scale data analysis.

 High-throughput screening methods are also used to characterize metabolic, pharmacokinetic and
toxicological data about new drugs. This HIT compound can be generated into LEAD compound.
 Principle of High Throughput Screening

 High throughput screening (HTS) involves the 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.

 Basically HTS is a process of screening and assaying large number of biological modulators and
effectors against selected and specific targets.
 Methods which are commonly followed are:

1) Target selection:

 Currently there are about 500 targets being used by companies.

 Cell membranes receptors, mostly G-protein coupled receptors make up the largest group (45% of the
total), Enzymes make up the next largest group (28%), followed by hormones (11%), unknowns (7%),
ion-channels (5%), nuclear receptors (2%), and finally DNA (2%).
2) HTS library to be screened:

 They usually consist of microtiter plates with frozen or dried samples of compounds to be screened.

 Initially the assays were carried out in 96-well plates but with advancement now there are also 1586-
well plates available.

 Typical HTS programs have potentials to screening up to 10000 compounds per day, while some
laboratories with Ultra High Throughput Screening (UHTS) can perform100,000 assays per day.
3) Assay design:

Assays mainly divided into biochemical and cell based assays.

Biochemical assay are further divided into two homogenous and heterogenous assays.

1) Homogenous assay:

i) Measurement are based on the distinct physical/chemical properties of analyte, or interaction

between analyte and surrounding environment.

ii) It is a single step process; reagent may be added at single stage or in multiple steps.

iii) It only involves usual steps like fluid addition, incubation and reading.

iv) It can be coupled with different detection technique fluorescence, radiometric for HTS.
Advantage:
i) Simplicity (Mix and read)
ii) Reduction of cost and robotic complexity.

2) Heterogenous assay:

i) Heterogeneous assays involves additional steps like filtration centrifugation. that

separates component(s) to be measured from the rest of component which may interfere
in assay.

ii) contributes to the high signal to background ratio.

Biochemical assays:
i) Biochemical assays are receptor, protein or enzyme based assays uses the particular target in a purified
form.
ii) Biochemical assays are most frequently carried out using scintillation proximity assay (SPA), radiometric,
colorimetric fluorescence detection techniques.
iii) Scintillation Proximity Assay is a technology whereby binding reactions can be assayed without the
washing or filtration procedures normally used to separate bound from free fractions.