High Throughput Screening drugg discovery.pdf

Commonly used terms in drug discovery
• High throughput screen: an optimized, miniaturized assay format that enables the testing of >
100,000 chemically diverse compounds per day.
• Assay: a test system in which biological activity can be detected
• Hit: a molecule with confirmed concentration-dependent activity in a screen, and known chemical
structure. The output of most screens
• Progressible hit: a representative of a compound series with activity via acceptable mechanism of
action and some limited structure-activity relationship information
• Lead: a compound with potential (as measured by potency, selectivity, physico-chemical properties,
absence of toxicity or novelty) to progress to a full drug development programme

Drug Discovery Process:
•The key steps of drug discovery are:
•Research – it takes average 2 to 3 years
•Pre-clinical testing - average 1 year
•Clinical trial testing (involving human
patients) - average 10 years
•Regulatory approval - average 2 years

High-throughput screening (HTS)
• High throughput screening (HTS) is the use of automated equipment to rapidly test
thousands to millions of samples for biological activity at the model organism, cellular,
pathway, or molecular level.
• HTS can identify active compounds, antibodies, genes, or one or more candidates
based on specific criteria.
• HTS uses robotics, data processing/control software, liquid handling devices, and
sensitive detectors to quickly conduct millions of chemical, genetic, or pharmacological
tests.
• It allows automation to quickly assay the biological or biochemical activity of a large
number of compounds.
• It is a useful for discovering ligands for receptors, enzymes, ion channels or other
pharmacological targets, or pharmacologically profiling a cellular or biochemical
pathway of interest
• It is a drug-discovery process widely used in the pharmaceutical industry.

❑ Steps in the HTS
• 1st stage screening
o To Test the optical clarity, abrasion resistance, and adhesion
o It Eliminates ~90% of samples
• 2nd stage screening
o It Test whether the ability, integrity, gloss, and surface smoothness
o It eliminates the ~10% of the samples
• There are 4 steps in HTS
o Preparation of samples and compound libraries
o Establishment of a method suitable for lab automation
o Configuration of a robotic workstation
o Acquisition and handling of data

❑Elements of the HTS: -
1) Chemical compound libraries: - this comprises the database of a samples that
are reacted with the target molecules. Typical libraries might include a range
of proteins or genes.
2) Suitable assay method: - this must me easy to replicate and suitable for
automated.
3) Robotic system: - it carries out all the automated process, it add the target
compound and wells, prepare the plates, add the reagents, set the reaction
conditions and prepare the samples for measurement.
4) Data anaylis system: - it takes all the results generated from the assays and
screens them for positive hits.

❑ Types of HTS: -
❑ The most widely used HTS techniques for PPIs include the: -
• two-hybrid assay
• affinity purification
• fluorescence polarization (FP)
• fluorescence resonance energy transfer (FRET).
❑ Types of high throughput assays: -
• Homogeneous assay
• Heterogeneous assays
• Biochemical assays
• Applications

• Genomics: - it is a Core Facility provides technical, instrument, and
professional development and next generation sequencing, gene
expression services, flow cytometry
• Protein Analysis: - a protein high throughput formulation (HTF)
platform is based on the use of microplates.
• DNA Sequencing: - it refers to the amount of DNA molecules read at
the same time. Technologies are now capable of sequencing many
fragments of DNA in parallel. This enables scientists to read hundreds
of millions of DNA fragments and generate more data, with less time
and costs than ever before.
• High-throughput technology can also be put to use in other areas
besides drug development.

Detection method in HTS
• Spectroscopy: - It is used as a tool for studying the structures of atoms and molecules
• Mass spectrometry: - it also called mass spectroscopy, analytic technique by which
chemical substances are identified by the sorting of gaseous ions in electric and
magnetic fields according to their mass-to-charge ratios.
• Chromatography:- it is a technique for separating the components, or solutes, of a
mixture on the basis of the relative amounts of each solute.
• Calorimetry: - it enables the continuous monitoring of samples for a prolonged period.
• X ray diffraction: it powders diffraction (XRD) is a rapid analytical technique primarily
used for phase identification of a crystalline material and can provide information on
unit cell dimensions
• Microscopy: - it is a technical field of using microscopes to view samples & objects that
cannot be seen with the unaided eye.
• Radioactive methods: -Radioactive methods of analysis are particularly useful for the
detection and determination of trace quantities of materials and for the measurement
of larger quantities in complicated systems.

History
• HTS was invented by Dr Gyula Takatsky in 1951, who machined 6
rows of 12 wells in Lucite to make the first microtiter plate.
• The microtiter plate has further grown to include standardized 96,
384, 1536 well formats, with additional 3072 well nanoplate
formats.
• “twenty eight plates are currently run daily on the Accuri C6 Hyper
Cytometer combination. We could run up to 40 plates in a standard
8 hour workday, over 12,000 comp

•High throughput screening
for drug discovery
•Why High throughput screening need arises ?
•FACT 1: recent understanding of disease mechanisms has
dramatically increased no. of protein targets for new drug treatment
•FACT 2: new technologies have increased the no. of drugs that can
be tested for activity at these targets.

❑ Goals of HTS
❑ The primary goal of HTS is to identify through compound library
screenings, candidates that affect the target in the desired way, so-called
“hits” or “leads”.
❑ This is usually achieved employing liquid handling devices, robotics, plate
readers as detectors, and dedicated software for instrumentation control
and data processing.
❑ HTS screen about 50,000 – 100,000 compounds per day

Explanation
• High-throughput screening is a method for scientific experimentation especially
used in drug discovery and is relevant to biology and chemistry. This process in
combination with robotics, data processing and control software, liquid
handling devices and sensitive detectors allows a researcher to quickly conduct
millions of chemical, genetic or pharmacological tests.
• High-throughput screening can rapidly identify active compounds, antibodies or
genes which modulate a particular bimolecular pathway. It can be considered -
a process in which batches of compounds are tested for binding activity or
biological activity against target molecules.
• High-throughput screening is a process of screening more compounds against
more targets per unit time, which should generate more hits, which in turn will
generate more leads, subsequently generating more products.
• Various technologies like high-throughput screening defined by the number of
compounds tested to be in the range of 10,000-100,000 per day, ultra high-
throughput screening is defined by screening more than 100,000 data point
generated per day. These two technologies play a vital role in drug discovery to
find new chemical compounds.

Which strategy is best for hit identification?
➢ When a target is identified, a decision has to be made about which chemicals to
screen, in order to identify potential lead compounds.
➢ There are two types of strategies best for hit identification
• Random screening :
o All possible drug molecules screened against target. This is simply not possible.
• Focussed screening :
o A limited number of compounds are pre-selected for screening.
Has proved successful as a hit generation strategy.
Useful when 3D structure of target is known (e.g. crystal structure of a receptor)

❑ Procedure: -
• High-throughput screening in drug discovery is used to screen :
• Novel biological active compounds
• Natural products
• Combinatorial libraries (Ex: peptides; chemicals)
• Biological libraries
• DNA chips
• RNA chips
• Protein chips
• Modern microplates for high-throughput screening assays are
performed in automation-friendly microtiter plates with a 96, 384,
1536 or 3456 well format.
• These wells contain experimentally useful matter, often an aqueous
solution of dimethyl sulfoxide (DMSO).

• For most drug discovery labs, the library collection has grown from
400,000 to 1 million or more compounds. The standard paradigms
used to screen these libraries have evolved to automated 384 wells or
higher density single compound test formats.
• Primary screen is designed to rapidly identify hits from compound
libraries. The goals are to minimize the number of false positives and
maximize the number of confirmed hits.
• Depending on the assay, hit rates typically range between 0.1 – 5 per
cent. This number also depends on the cutoff parameters set by the
researchers, as well as the dynamic range of a given assay.
• Primary screens are run in multiplets of single compound
concentrations. Hits are then retested, usually independently from the
first assay.

• If a compound exhibits the same activity, it is coined as confirmed hit, which
proceeds to secondary screens or lead optimization. The results from lead
optimization are used to decide which substances will make it on to clinical
trials.
• In combination with bioinformatics, it allows potential drugs to be quickly and
efficiently screened to find candidates that should be explored in more detail.
Initial screening of these compounds for their binding ability is the job for high-
throughput screening.
• The key to high-throughput screening is to develop a test, or assay, in which
binding between a compound and a protein causes some visible change that
can be automatically read by a sensor. Typically the change is emission of light
by a fluorophore in the reaction mixture.
• One way to make this occur is to attach the fluorophore to the target protein in
such a way that its ability to fluoresce is diminished (quenched) when the
protein binds to another molecule. A different system measures the difference
in a particular property of light (polarization) emitted by bound versus unbound
fluorophores. Bound fluorophores are more highly polarized and this can be
detected by sensors.

❑ Procedure: -
• The sets of compounds produced by combinatorial chemistry are generally referred
to as libraries, which depending on how the solid- phase is handled, may be either
mixtures or individual compounds.
• There are a range of options for testing the libraries in a biological assay.
o Test mixture in solution
o Test individual compounds in solution Test compounds on the beads

Test mixture in solution :
• All the compounds are cleaved from the beads and tested in solution.
• If activity in a pharmacological screen is observed,
it is difficult to find out which compounds are active. To identify the
most active component, it is necessary to resynthesize the
compounds individually and thereby find the most potent. This
iterative process of resynthesis and screening is one of the most
simple and successful methods for identifying active compounds
from libraries.

Test individual compounds in solution
• A second method is to separate the beads manually into
individual wells and cleave the compounds from the solid-phase.
These compounds can now be tested as individual entities.
•Test compounds on the beads :
• A third method for screening is testing on the beads, using a
colorimetric or fluorescent assay technique. If there are active
compounds, the appropriate beads can be selected by color or
fluorescence.

Chromatography
• Chromatography (from Greek word ‘chroma’ a “color” and ‘graphic’ to “write”)
chroma mans a color and graphic means to write.
• It is the collection term for a set of laboratory techniques for the separation of
mixtures.
• The mixture is dissolved in a fluid called the mobile phase, which carries it
through a structure holding another material called the stationary phase.
• The various constituents of the mixture travel at it different speed causing them
to separate.
• The separation is based on differential partitioning between the mobile and
stationary phase.

➢ Stationary phase: -
• Stationary phase in chromatography is the one which does not move with the sample
whereas mobile phase in chromatography is one which moves with the sample.
• The stationary phase is polar, usually using silica.
• The stationary phase is composed of either a solid or liquid substance attached to a
glass or a metal surface.
➢ Mobile phase: -
• In chromatography, the mobile phase is a liquid or gas that flows through a
chromatographic column.
• The mobile phase carries the components of a mixture with it, and different
components travel at different rates.
• The mobile phase can be:
o A liquid solvent or mixture of solvents
o A chemically inert gas
o A liquid phase coated on the surface of a solid phase

History of chromatography
• It was first employed by the Russian Italian botanist scientists (Mikhail Tsvet) in 1900.
• He continuous to work in the chromatography in the first decade of the 20th century
,primarily for the separation of plant pigment such a ‘chlorophyll’ (which is green) and
‘carotenoids’ (which are orange and yellow).
• The new forms of chromatography developed in the 1930s and 1940s made the technique
useful for a wide range of separation processes and chemical analysis tasks, especially in
biochemistry.
➢ Uses: -
• Chromatography is a method that is used in laboratories for the separation of a mixture. It
is used to test drug levels and water purity.
• It is also used to determine the nutritional value of the food sample.
• It is used to determine the type of chlorophyll in various photosynthetic organisms.
• Pharmaceuticals: Used to analyze components in water, raw materials, and products
• Food and beverage: Used for food and beverage testing
• Molecular biology: Used for DNA fingerprinting and bioinformatics
• Drug testing: Used for drug testing
• Forensics: Used for evidence analysis

➢ Principles: -
• Chromatography is usually consists of a mobile phase and stationary phase.
• Chromatography can be used for the purification or analysis of components from a mixture,
including simple and complex molecules.
• The interaction between the mobile and stationary phase results in the separation of a
compounds from a mixture.
• Different types of chromatographic techniques, such as liquid chromatography (LC), are used
for the separation of proteins.
➢ Applications: -
• Separation:- Chromatography can separate different colors of ink, amino acids, proteins, and
more
• Identification:- Chromatography can identify and separate preservatives and additives in
food
• DNA fingerprinting:- Chromatography can be used in DNA fingerprinting and bioinformatics
• Ion exchange: -Ion exchange chromatography can separate organic and inorganic ions from
an aqueous solution
• Affinity:-Affinity chromatography separates a sample based on its molecular bonds

➢ Types of chromatography: -
• There are several types of chromatography are: -
1. Paper chromatography
2. Thin layer chromatography
3. High - performance liquid chromatography
4. Column chromatography
5. Ion exchange chromatography
6. Gas liquid chromatography
7. Gel chromatography
8. Affinity chromatography

1. Paper chromatography: - it is an analytical chemistry technique that separates
dissolved chemical substances based on their migration rates across sheets of paper.
2. Thin-layer chromatography (TLC): - it is a technique used to separate mixtures of
compounds based on differences in polarity.
3. High-performance liquid chromatography (HPLC) : - it is an analytical technique used
in analytical chemistry and biochemistry to separate, identify, and quantify
components in a mixture.
4. Column chromatography: - it is a separation technique used to isolate compounds
from complex mixtures.
5. Ion exchange chromatography: - Ion exchange chromatography (IEC) is a technique
for separating compounds based on their net charge
6. Gas–liquid chromatography (GLC): - it is a commonly used method for lipid analysis.
7. Gel chromatography: - it is a technique in analytical chemistry that separates
chemical substances by how quickly they pass through a porous, semisolid substance.
Also known as gel filtration
8. Affinity chromatography: - it is a liquid chromatographic method that separates a
biomolecule from a mixture.

1. Paper chromatography: - it is an analytical chemistry technique that separates dissolved
chemical substances based on their migration rates across sheets of paper.
➢ Principle of Paper chromatography: -
• Paper chromatography is of two types based on two different principles.
• The first is the paper adsorption chromatography that is based on the varying degree of
interaction between the molecules and the stationary phase.
• The second type of paper chromatography is the paper partition chromatography.
• It is based on the principle that the moisture on the cellulose paper acts as a stationary
phase for the molecules moving with the mobile phase.
➢ Steps of Paper chromatography: -
• The stationary phase is selected as a fine quality cellulosic paper.
• Different combinations of organic and inorganic solvents are taken as the mobile phase.
• The sample loaded paper is then carefully dipped into the mobile phase not more than
the height of 1 cm
• After the mobile phase reaches near the edge of the paper, the paper is taken out.

➢ Uses of Paper chromatography: -
• Paper chromatography is performed to detect the purity of various pharmaceutical
products.
• It can also be employed to detect contamination in various samples, like food and
beverages.
• This method can also be used for the separation of impurities from various industrial
products.
2. Thin-layer chromatography (TLC): -
• it is a technique used to separate mixtures of compounds based on differences in polarity.
• It's a widely used separation technique for quantitative and qualitative analysis.
➢ Principle of Thin-layer chromatography (TLC): -
• The substrate/ ligand is bound to the stationary phase so that the reactive sites for the
binding of components are exposed.
• After separation, the molecules are seen as spots at a different location throughout the
stationary phase.
• The detection of molecules is performed by various techniques.

➢ Steps of Thin-layer chromatography (TLC): -
• The stationary phase is uniformly applied on the solid support (glass, thin plate or
aluminum foil) and dried.
• The sample is injected as spots on the stationary phase about 1 cm above the edge of
the plate.
• The sample loaded plate is then carefully dipped into the mobile phase not more than
the height of 1 cm.
➢ Uses of Thin-layer chromatography (TLC): -
• Thin-layer chromatography is routinely performed in laboratories to identify different
substances present in a mixture.
• This technique helps in the analysis of fibers in forensics.
• TLC also allows the assay of various pharmaceutical products.

3. High-performance liquid chromatography (HPLC): -
• High-performance liquid chromatography (HPLC) is an analytical technique used in
analytical chemistry and biochemistry to separate, identify, and quantify components in
a mixture.
• It was first discovered in the early twentieth century and was initially used to separate
colored compounds.
➢ Principle of HPLC: -
• The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
• However, the molecules with lower affinity move with a faster movement, thus allowing
the molecules to be separated in different fractions.
➢ Steps of HPLC
• The column is prepared by taking a glass tube that is dried and coated with a thin,
uniform layer of stationary phase (cellulose, silica).
• The mobile phase then moves down to a detector that detects molecules at a certain
absorbance wavelength.
• The separated molecules can further be analyzed for various purposes.

➢ Uses of HPLC
• High-performance liquid chromatography is used in the analysis of pollutants present
in environmental samples.
• It is performed to maintain product purity and quality control of various industrial
productions.
• This technique can also be used to separate different biological molecules like
proteins and nucleic acids.
❑ Formula (reflective formula) paper chromatography
RF = distance travelled by the substance
distance travelled by the solvent
• The RF value helps for the identification of unknown

Characteristics and properties of therapeutic area
• A therapeutic target is a biological molecule, biological pathway, or physio local response
that is associated with a particular disease process.
• It may be inhibited or activated by a therapy in a way that will change the course of the
disease in a positive way.
• Therapeutic area mean, the grouping of similar diseases 0or conditions under a
generalized heading.
• E.g.- oncology
- cardiology
- dermatology
- neurology
- hematology
• These therapeutic areas guide the efforts of researchers, physicians and pharmaceutical
companies in their pursuit of better healthcare solutions.
➢ Cardiovascular (heart disease)
• The heart disease describes a range of conditions that affect the heart
- blood vessels disease
- irregular heartbeats (arrythmia)
- heart problems (congenital heart defects)
- disease of the heart muscle
- heart valve disease

➢ Symptoms: -
• Angina or chest pain
• Difficulty in breathing
• Fatigue
• Swelling due to fluid retention or edema
➢ Causes: -
• Damage to all or parts of the heart
• A low supply of oxygen and nutrients to heart
• The rhythm of the heart
➢ Risk factors: -
• High blood pressure (BP)
• High cholesterol
• Smoking
• A high intake of alcohol
• Diabetes
• Sleep apnea

Properties of therapeutic area

• The patient should be the primary focus of interaction
• A professional attitude sets the tone of the therapeutic relationship
• Use self-disclosure cautiously & only when it has a therapeutic purpose
• Avoid social relationship with patients & maintain confidently
• Implement interventions on theoretical basis
• Guide the patient to reinterpret his or her experience rationally
Characteristics of therapeutic area

Question bank
1. Write the relationship between drug targets and therapeutic drugs?
• The protein molecules are the primary target of drug molecules.
• Molecular biology is providing new insights into the nature of genes, proteins and the
relationship between them.
• Whereas the time-honored biochemical and physiological approaches can show how
disease affects function at the level of cells, tissues, organs and individuals.
• The links between the two nevertheless remain tenuous, fact which greatly limits our
ability to relate drug targets to therapeutic effects.
2. Write a short note on therapeutic targets?
• A therapeutic target is a biological molecule, biological pathway, or physio local response
that is associated with a particular disease process.
• It may be inhibited or activated by a therapy in a way that will change the course of the
disease in a positive way.
• Therapeutic targets are used to screen potential therapies in the discovery phase of the
therapy development process.
• the pathway information and the corresponding drugs/ligands directed at each of these
targets.
• Pharmaceutical agents generally exert their therapeutic effect by binding to a particular
protein or nucleic acid target

3. Write advantages and disadvantages of a small molecule drug?
❑ Advantages: -
• ‘Chemical space’ is so vast that synthetic chemicals according to many experts, it have the
potential for a right molecule exists: it is just a matter of finding it.
• Doctors and patients are thoroughly familiar with conventional drugs as medicines, and
the many different routes of administration that are available.
• Clinical pharmacology in its broadest sense has become part of the knowledge base of
every practicing doctor and indeed part of everyday culture.
• Oral administration is often possible, as well as other routes where appropriates.
❑ Disadvantages: -
• Side and effects and toxicity remain a serious and unpredictable problem.
• It causing failures in late development, or even after registration.
• Humans and other animals have highly developed mechanism for eliminating foreign
molecules.
• The drug design often has to contend with pharmacokinetic problems.
• Oral administration is poor for many compounds and peptides cannot be given orally.

4. write a short notes on conventional therapeutic drugs?
• The conventional drug design methods include random screening of chemicals found in
nature or synthesized in laboratories.
• The time consuming is 10 – 15 years and it is very expensive.
• Small-molecule drugs, either synthetic compounds or natural products have for long
bene the mainstay of therapeutics.
• The pre-eminent role of conventional small-molecule drugs may decline as
biopharmaceutical products Grow in importance
• The drugs delivery system will allow the drugs to act much more selectivity where they
are needed, and thus reduce the burden of side effects.

High Throughput Screening drugg discovery.pdf

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