1

Protein Microarray—  The concept and methodology of protein microarrays was first
introduced and illustrated in antibody microarrays (also referred to as antibody matrix) in 1983 in a
scientific publication

A protein microarray (or protein chip) is a high-throughput method used to track the interactions

and activities of proteins, and to determine their function, and determining function on a large scale.

The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead,
or microtitre plate, to which an array of capture proteins is bound.

 Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction
between the probe and the immobilised protein emits a fluorescent signal that is read by a laser
scanner.

Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small
quantities of samples and reagents.

Classified into two broad categories

According to the immobilized molecules (antigen or antibody)

Forward phase protein microarrays Reverse phase protein microarrays

 2

In protein microarray experiments, signals can be detected by label-based or label-free strategies. Mass
spectrometry (MS) has played a central role in protein detection.

Application of Protein Microarray—

Protein microarrays are potentially powerful tools in biochemistry and molecular biology. They provide a powerful tool to
profile protein–protein interactions in high-throughput and to quantify the abundances and posttranslational modifications of
different kinds of proteins in complex mixtures.

Potential applications of
protein microarray in
biochemistry and
molecular biology. It can
be used for biomarker
identification from a
number of different
diseases by profiling
candidates present in
patients and healthy
control subjects. By using
the small molecule
profiling application, it
can also be an ideal drug
discovery tool to identify
drug targets and
understand their
mechanisms of action.
Besides, it can be
performed for functional analysis to identify protein-protein interactions and substrates or enzymes in
posttranslational modification.

1. Biomarker screening—
Protein microarray has been successfully used for biomarker to identification from a number of
different diseases including but not limit to colon cancer, breast carcinoma,prostate cancer,
bladder cancer, and non-small cell lung cancer.
SELDI-TOF-MS protein microarrays have been a high-throughput technique for analysis of
complex biological specimens for biomarker profiling. They have been successfully applied to
identify serum biomarkers for cancers as a simple, sensitive and highly reproducible method.
2. Drug Discovery—

Protein microarrays have also been used in drug discovery for target identification and validation. In
2004, Schreiber and co-workers described for the first time the use of a protein microarray for high-
throughput screening of small molecules.
 3

3. Enzyme substrate profiling—

protein microarrays technology is an excellent method to identify enzyme substrates, especially
the profiling of kinases, ubiquitin ligases, and methyltransferases for target discovery and
validation
Identification of protein targets of post-translational modification is an important analytical
problem in biological field.
Protein microarrays exposed to cellular extracts that offers a rapid and convenient means of
identifying modified protein .
This technique allows researchers to rapidly assess the specificity of drugs on enzymes.
4. Small molecule profiling—
Protein microarrays can also be an ideal drug discovery tool for small molecules. Researchers
using the small molecule profiling application on the protein microarrays have been able to
identify drug targets and understand their mechanisms of action.
Small molecules possess the ability to interact with proteins and perturb their specific
function.commonly utilized mass spectrometry-based approaches for identifying the target
proteins for a small molecule have a number of limitations, particularly in terms of throughput
and time and resource consumption.
5. Protein-protein interaction—
In order to uncover new protein–protein interactions, protein microarrays can be used to profile
the certain purified protein against functional proteins and identify the possible binding
partners.
Using protein microarrays, expanded the substantial interactome of the NF-kB essential
modulator. They identified a total of 112 protein interactors, in which more than 30% were
kinases, while at least 25% were involved in signal transduction.
6. Antibody specificity profiling—

DNA Microarray Protein microarray

Two dimensionally arranged series of DNA Arranged of purified proteins at high spatial
nucleotide also known as probes on solid surface density on glass slide.
like glass slide or silicone thin film used.
The probes such as cDNA or cRNA Probes such as antibody labelled
Another important application of protein microarrays has been to determine the specificity of
antibodies.
Antibodies are the mostwidely used protein ligand for all proteins in the human and other
proteomes. Antibody specificity is a key feature for determining an antibody’s value and it is
important for research and therapeutic antibody development. With these considerations in
mind, a new protein binding ligand called a synthetic antibody or synbody has been developed,
which is composed of two peptides linked by a scaffold and then the synbody was screened
 4

against a library of proteins to discover the target. This method has delivered a high-affinity
ligand that specially binds a target protein in a single discovery step.

Antisense Technologies—
Antisense technology is a tool that is used for the Inhibition of gene expression.
Antisense technologies are a suite of techniques that, together, form a very powerful tool
for studying gene function (functional genomics) and for discovering new and more specific
treatments of diseases in humans, animals and plants (antisense therapeutics.
 5

Antisense refers to the laboratory manipulation and/or modification of DNA or RNA so that its
components (nucleotides) form a complementary copy of normal, or “sense,” messenger RNA
(mRNA).
The binding, or hybridization, of antisense nucleic acid sequences to a specific mRNA target will,
through a number of different mechanisms, interrupt normal cellular processing of the genetic
message of a gene.
This interruption, sometimes referred to as “knockdown” or “knock-out” depending upon
whether or not the message is either partially or completely eliminated.

In Antisense technology, synthetically – produced complementary molecules seek out and bind
to messenger RNA (mRNA), blocking the final step of protein production. mRNA is the nucleic
acid molecule that carries genetic information from the DNA to the other cellular machinery
involved in the protein production. By Binding to mRNA, the antisense drugs interrupt and inhibit
the production of specific disease-related proteins .
“Sense” refers to the original sequence of the DNA or RNA molecule. “Antisense” refers to the
complementary sequence of the DNA or RNA molecules.

THE BASICS OF ANTISENSE—

A Sense strand is a 5’ to 3’ mRNA molecule or DNA molecule. The complementary strands or
mirror strand to the sense is called an antisense.
Antisense technology is the process in which the antisense strand hydrogen bonds with the
targeted sense strand. When an antisense strand binds to a mRNA sense strand, a cell will
 6

recognize the double helix as foreign to the cell and proceed to degrade the faculty mRNA
molecule thus preventing the production of undesired protein.
Although DNA is already a double stranded molecule, antisense technology can be applied to it
building a triplex formation.
RNA antisense strands can be either catalytic, or non catalytic .The catalytic antisense strands,
also called ribozymes, which will cleave the RNA molecule at specific sequences. A Non catalytic
RNA antisense strand blocks further RNA processing, i.e. modifying the mRNA strand or
transcription.
Technology Working—
The therapeutic objective of antisense technology is to block the production of disease
technology is to block the production of disease causing proteins. This is achieved by creating a
synthetic “antisense” or complementary nucleotide sequence of DNA or RNA that interacts with,
and binds to the “sense” or original mRNA sequence. This “mRNA”- antisense complex” can no
longer be translated and the disease causing protein cannot be produced.

Several theories include:

That the dsRNA prevents ribosome’s from binding to the sense RNA and translating .
The ds-RNA cannot be transported from within the nucleus to the cytosol. This is where
translation occurs .
That ds-RNA is susceptible to endoribonucleases that would otherwise not affect single
stranded RNA, but degrade the ds-RNA.
Cloning of aRNA—
In order for aRNA to black translation, it has to be inserted into the proper cells so it can bind to its
complementary sense strand. There are several ways to incorporate aRNA into a cell. One method is to
create a plasmid that codes for the aRNA. In essence, this is a plasmid that has a promoter that initiatives
transcription in the direction of the 3’ends of the sense stand to the 5end. Which corresponds to 5’ to 3’
transcription of the antisense strand? Next, this liposome’s can be used to transfect target cells with the
newly formed plasmids. Liposomes are cheap and efficient for this task5 . Adenoviruses can also be used
to infects cells and delivers aRNA sequences. This method has higher transduction efficiency than liposome. A
 7

northern blat can be used to detect whether the aRNA is produced within the cells, and a western blat can be used to
measure amount of the mRNA gene that is expressed in wild type and mutant cells with.
RNA Inhibition—
Inserting Antisense into cells
a. Endocytosis: One of the simplest methods to get nucleotide in the cell, it relies on the cells
naturalprocess of receptor mediated endocytosis. The drawbacks to this method are the
long amount of time for any accumulation to occur, the unreliable result, and the
inefficiency.
b. Micro-Infection: As the name implies, the antisense molecule would be injected into the
cell. The yield of this method is very high, but because of the precision needed to inject a
very small cell with smaller molecules only about 100 cells can be injected per day.
c. ) Liposome–Encapsulation: This is the most effective method, but also a very expensive
one23. Liposome encapsulation can be achieved by using products such as lipofect ACE™ to
create a cationic phospholipids bilayer that will surround the nucleotide sequence. The
resulting liposome can merge with the cell membrane allowing the antisense to enter the
cell.
d. Electroporation: The conventional method of adding a nucleotide sequence to a cell can
also be used. The antisense molecule should transverse the cell membrane offer a shock is
applied to the cells23 .
e. Antisense PG gene: The PG enzyme is responsible for the breakdown pectin. Pectin is a
building block in cell walls, and is what gives tomatoes their firmness.

Triplex Antisense Technology—

This triplex technology provides the opportunity to reduce gene transcription itself rather than to
destroy mRNA once it is produced. Because the triplex oligonucleotides can be made to permanently
after the DNA after localizing to specific target sites, the technology actually has the potential to
permanently silence genes.

Application—
Cholesterol therapy—

Physiologically, cholesterol is a structural component of cell membranes, as well as serving as a

precursor of steroid hormones, a precursor of vitamin D, and a precursor of bile in the liver. Other than
benefits cholesterol participates in pathological processes involved in formation of coronary
atherosclerotic plaques, as pure cholesterol crystals form components in these lesions. As apoB
comprises VLDL, IDL, and LDL, targeting apoB mRNA has combined impact on atherogenic disorders,
even in the most severe genetically-regulated disease; e.g., familial hypercholesterolemia. The apoB
protein levels increase the burden the of atherogenic proteins .

ISIS Pharmaceuticals is currently developing mipomersen, an antisense oligonucleotide

targeting apoB. At present, mipomersen has reached the Phase III clinical trial in patients with
severe hyperlipidemia who are resistant to statins
 8

Cancer therapy—

The biggest problems of standard anticancer therapies are their side effect profiles, with patients
experiencing life-threatening complications. In contradistinction, an antisense technology with its very
selective targeting of mRNA provides a unique strategy for blocking resistant cancer cells with maximum
efficacy and minimum side effects. An antisense strategy also may be used to target proteins responsible
for development of resistance to drugs.

e.g. OGX-427 Heat shock protein 27 Phase I/II Prostate, ovarian, non-small-cell lung, breast, bladder
cancers (with docetaxel).

Duchenne muscular dystrophy—

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular X-recessive
disorder. DMD is caused by the absence of dystrophin protein as a consequence of different mutations
in the dystrophin gene (DMD gene), resulting in nonfunctional dystrophin proteins. Since dystrophin is
required for muscle fiber membrane stability during contraction, its loss leads to permanent muscle
fiber damage. In recent years, an exon-skipping strategy by antisense oligonucleotides has been tried to
restore dystrophin expression at the sarcolemma. The antisense 2 ′- O-methyl and morpholinos
oligonucleotides are designed individually for the mutated DMD mRNA to change its splicing pattern and
promoting functional translation.

Antisense Oligoucleotides—

short, single-stranded DNA molecules that interact with messenger RNA to prevent translation of a

targeted gene. Their DNA sequence is complementary to the specific mRNA target; binding leads to
degradation of the DNA sequences with failure of protein production .

This blocks the ability of the RNA to make a protein or work in other ways. Antisense oligonucleotides
may be used to block the production of proteins needed for cell growth. They are being studied in the
treatment of several types of cancer. Also called antisense agent.
 9

SiRNAs—

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is

a class of double-stranded RNA non-coding RNA molecules, 20-25 base pairs in length, similar
to miRNA, and operating within the RNA interference (RNAi) pathway. t interferes with
the expression of specific genes with complementary nucleotide sequences by degrading mRNA
after transcription, preventing translation.

Zinc finger proteins— The “finger” refers to the secondary structures (α-helix and β-sheet) that
are held together by the Zn ion.

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are
extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional activation,
regulation of apoptosis, protein folding and assembly, and lipid binding.

A new group of transcriptional activator proteins with a 30 amino acid repeating region. This new class
of proteins was able to bind specific sequences of DNA.

The zinc finger structure is maintained by the zinc ion, which coordinates cysteine and histidine in
different combinations.

In classical C2H2 zinc-finger proteins, two cysteines in one chain and two histidines in other one are
coordinated by a zinc ion.

Crystallographic studies revealed that classical zinc-finger domains have two β-sheets and one α-helix.

Non-classical types of zinc-finger differ in cysteine/histidine combinations, such as C2–H2, C2–CH, and
C2–C2.

The most important and abundant types of zinc-finger domain proteins include C2H2, really interesting
new gene (RING), plant homeodomain (PHD).
 10

PHYSIOLOGICAL ROLE OF ZNFS—

1. Skin— It have ability to regulate gene expression, ZNF proteins participate in numerous
physiological processes, including cell proliferation, differentiation, and apoptosis, thereby
maintaining tissue homeostasis .

zinc-finger protein KLF4(C2H2-type, transcription factor) that has a crucial role in keratinocyte
differentiation, is the transcription factor. in the epidermis, KLF4 is mainly expressed in
suprabasal layers, where it modulates the expression of genes involved in keratinocyte
differentiation.

2. Intestine— ZNF proteins are also involved in intestinal epithelium biology. KLF4 also has a key
role in the intestines. In this tissue, KLF4 is expressed in the terminally differentiated epithelial
cells (luminal surface) and goblet cells (crypts), where it promotes differentiation and inhibits
proliferation. The GATA family members GATA4 (GATA-type, transcription factor) and GATA6
(GATA-type, transcription factor) have important roles in differentiation and homeostasis of the
small intestinal epithelium.
3. Muscle— ZNFs have a regulatory function in muscle differentiation. For example, SET and
MYND Domain Containing 1 (SMYD1), which is specifically expressed in striated muscle, acts as
an essential regulator of myogenesis.
4. Adipose tissue— Recent studies revealed an increased number of ZNFs as key transcriptional
regulators involved in adipogenesis. ZNF638 promotes adipogenesis by acting as a
transcriptional cofactor of CCAAT/enhancer-binding protein (C/EBP) and results inthe expression
of peroxisome proliferator-activated receptor γ (PPARG), which regulates adipocyte
differentiation.

ROLE OF ZNFS IN DISEASES—

1. Tumour suppressor and oncogenic functions of ZNFs— Recent findings have highlighted the
importance of ZNFs in cancer onset and progression.
 11

The zinc-finger family includes both tumour suppressor genes and oncogenes. ZNFs are involved in
all the principal pathways of cancer progression from carcinogenesis to metastasis formation.

ZNF281 (C2H2-type) in tumorigenesis and tumour invasion. ZNF281 is involved in two crucial
processes in cancer: the DNA damage response (DDR) 62–65 and the epithelial–mesenchymal transition
(EMT). ZNF281 expression is increased upon DNA damage induced by drugs in several cancer types.

2. Neurodegenerative diseases— ZNFs have been demonstrated to have an important role in the
pathogenesis of neuronal diseases.Spinal muscular atrophy (SMA) is a rare neuromuscular
disorder characterized by loss of α-motor neurons in the anterior horn of the spinal cord and
progressive muscle wasting, often leading to early death. The cause of the disease is a mutation
in the Survival Motor Neurons 1 (SMN1) gene that that results in reduced expression of the full-
length SMN protein, which is necessary for survival of motor neuronsresults in reduced
expression of the full-length SMN protein, which is necessary for survival of motor neurons.
ZPR1 mutation leads to this disease. ZNF746 (C2H2-type, not transcription factor), also known as
Parkin Interacting Substrate(PARIS), has been identified in the pathogenesis of Parkinson’s
disease (PD).
3. Psoriasis— ZNF750. Increasing evidence confirms the important roles of ZNFs in psoriasis.
Psoriasis is a chronic inflammatory disorder of the skin, which varies in severity and clinical
manifestation. ZNF750 is associated with a seborrhea-like dermatitis with psoriasiform
elements. ZNF750 has been identified in psoriasis patients and results in a frameshift mutation.
This mutation leads to the production of a truncated protein that does not contain the zinc-
finger domain. Downregulation of ZNF750 leads to reduced expression of genes involved in
epidermal differentiation and skin barrier formation

Role Of Transgenic animals— See in previous Documents

High Throughput Screening—

o HIGH THROUGHPUT SCREENING (HTS) is identification of one or more positive
candidates extracted from a pool of possible candidates based on specific criteria. It is a drug-
discovery process widely used in the pharmaceutical industry.

ž DETECTION METHODS IN HTS:

• Spectroscopy

• Mass Spectrometry

• Chromatography

• Calorimetry

• X-ray diffraction

• Microscopy
 12

• Radioactive methods

o SPECTROSCOPY IN HTS:

• Fluorescence Spectroscopy

• Total internal reflection fluorescence (TIRF)

• Nuclear magnetic resonance (NMR)

• Absorption and luminescence

• Fourier transformed infrared(FTIR)

• Light scattering

o CHROMATOGRAPHY IN HTS:

• Gas chromatography (GC)

• Thin layer chromatography

• Liquid chromatography (HPLC)

• Ion Exchange chromatography

• Reverse phase chromatography

• Hydrophobic interaction chromatography

• Affinity chromatography

o CALORIMETRY IN HTS:

• Isothermal titration Calorimetry (ITC)

• Differential scanning Calorimetry (DSC)

o MICROSCOPY IN HTS:

• Scanning Tunnelling Microscopy

• Atomic Force Microscopy

• Confocal Microscopy

o Uses:

To screen Micro arrays such as:

 13

• DNA chips

• RNA chips

• Protein chips

• To screen for all kind of novel biological active compounds

• Natural products

Methodology—

o The heart of the HTS system is a plate, or tray, which consists of tiny wells where assay reagents
and samples are deposited, and their reactions monitored.

o The configuration of the plate has changed from 96 wells (in a matrix of 8 rows by 12 columns)
to 384, and now to a high - density 1536 - well format, which enables large - scale screening.

o Assay reagents may be coated onto the plates or deposited in liquid form together with test
samples into the well.

o Both samples and assay reagents may be incubated, and those that interact show signals, which
can be detected .

o The aim of HTS and UHTS is cost effectiveness and speed of compound scanning.
 14

o Cell - based assays have become an important test compared with other in vitro assays, as they
can provide information about bioavailability, cytotoxicity and effects on biochemical pathway

o The enzyme - based and cell - based assay systems consist of receptors or mimetics of receptors
(components that mimic active parts of receptors)

o Normally the assays are linked to an indicator that shows the ligands – receptor interaction as
some form of signal

The advantage of cell - based assays over biochemical assays is that cell - based assays enable
the analysis of sample compound activity in an environment that is similar to the one in which

a drug would act. It also provides a platform for toxicity studies.

 15

Cell Based Assay—

Cell-based assays refer to any of a number of different experiments based on the use of live cells

This is a general definition and can include a variety of assays that measure cell proliferation, toxicity,
motility, production of a measurable product and morphology

Cell-based assays offer a more accurate representation of the real-life model since live cells are used.

o FOUR KEY ELEMENTS OF CELL BASED ASSAY:

• A cellular component e.g. a cell line or a primary cell population

• A target (substrate) molecule that records the cellular response

• An instrument to conduct and monitor the assay.An informatics component to manage and
analyse data from the assay .
 16

Cell based assays advantages over Biochemical assays—

o Assays do not require purification of the target protein

o Can immediately select against compounds/ potential drugs that are generally cytotoxic, or that
cannot permeate cellular membranes to reach intracellular sites

o Hit/lead compounds identified by cell based assays have passed important validation steps,
saving time and costs in drug development

o Cell-based assays visualize all possible drug-target interactions e.g. activators, target
interactions .

DISADVANTAGE OF HTS

o High cost

o Low data quality

o Local contamination

o Need of relatively pure produuct

 17

APPLICATIONS—

• High-throughput technology can also be put to use in other areas besides drug development.

1- Genomics Applications

DNA Sequencing

• Chemical genomics involves generating large collections of small molecules and using them to
modulate cellular states .

• gene expression–based high-throughput screening (GE-HTS) in which a gene expression

signature is used as a surrogate for cellular states.

• its application in a particular setting: the identification of compounds that induce the
differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8
that reliably induced the differentiation signature

2- Protein Analysis

• HTS systems to mimic chromatographic purification steps was established, several studies were
performed successfully including scale down purification.

• Here, we propose a method for studying different purification conditions that can be used for
any recombinant protein, including complex and glycosylated proteins, using low binding filter
microplates

3-In drug discovery

• High-throughput screening (HTS) is one of the newest techniques used in drug design and may
be applied in biological and chemical sciences.Characterization of metabolic,Pka and
toxiological data about new drugs.

• This method, due to utilization of robots, detectors and software that regulate the whole
process, enables a series of analyses of chemical compounds to be conducted in a short time
and the affinity of biological structures which is often related to toxicity to be defined.

4-Systematic study of mitochondrial toxicity of enviromental chemicals

-Recent decades have seen a rapid increase in reported toxic effects of drugs and pollutants on
mitochondria. Researchers have also documented many genetic differences leading to mitochondrial
diseases, currently reported to affect ∼1 person in 4,300, creating a large number of potential gene-
environment interactions in mitochondrial toxicity

• High throghput screening of dendrimer-binding drugs

• To fractionate plant natural products for drug discovery

 18

• To screen for all kind of novel biological active comounds

-natural products

-combinatorial libraries(peptides,chemicals)

-biological libraries

In silico lead discovery techniques—

Drug discovery process is a critical issue in the pharmaceutical industry since it is a very costly and time
consuming process to produce new drug potentials and enlarge the scope of diseases incurred. There
are many factors responsible for the failure of different drugs such as lack of effectiveness, side
effects, poor pharmacokinetics, and marketable reason.

Two different methods are widely used in the pharmaceutical industry for finding hits are—

1. High throughput screening

2. Virtual screening

High throughput screening

The chemical compounds are synthesized, and screened against protein based or cell based assays.

This process is commonly used in all major pharmaceutical industries.

However, the cost in synthesis of each compound, in vitro testing and low hit rate are posing huge
problems for pharmaceutical industries.

Current efforts within the industry are directed to reduce the timeline and costs. Besides

HTS campaigns to identify compounds exerting a desired phenotype or entire pathways, many of these
drugs are failing in clinical development either because of poor pharmacokinetic characteristics or to
intolerable side effects, which may reflect insufficient specificity of the compounds.

In view of the above problems in finding new drugs by HTS; cost effective, reliable virtual screening
procedures are in practice. The so-called in silico approaches, using computational environments as their
experimental laboratories.

DRUG DISCOVERY—

Drugs are chemicals that prevent disease or assist in restoring health to diseased individuals..
 19

Drug discovery is one of the most crucial components of the pharmaceutical industry's Research and
Development (R&D) process and is the essential first step in the generation of any robust, innovative
drug pipeline . The process of drug development aims towards the identification of compounds with
pharmacological interest to assist in the treatment of diseases and ultimately to improve the quality of
life. The compounds used in pharmacology are mainly small organic molecules (ligands) which interact
with specific biomolecules (receptors).

Traditional Drug Discovery Limitations—

1. In the distant past, designing a new drug by changing the molecular structure of an existing drug
was a slow process of trial and error.
2. Time consuming, multistep process.
3. Must be include the animals to determine the ADME & their toxicity.
4. Development process has resulted in high attrition rates with failures attributed to poor
pharmacokinetics (39%), lack of efficacy (30%), animal toxicity (11%), adverse effects in humans
(10%) and various commercial and miscellaneous factors.

Drug Design—

Drug design, sometimes referred to as rational drug design (or more simply rational design), is the
inventive process of finding new medications based on the knowledge of biological targets.

Rational drug design can be broadly divided into two categories—

Development of small molecules with desired Development of small molecules

Properties toward targets, biomolecules with predefined properties toward
(Proteins or nucleic acids), whose functional targets, whose cellular functions and
roles in cellular processes and 3D structural their structural information may be
Information are known. be known or unknown.

Drug designing—

Selected/designed molecule should be—

 Organic small molecule

 Complementary in shape to target.
 Oppositely charged to the biomolecular target.

This molecule should be—

 Interact with target.

 Bind to the target. .Activates or inhibit the target(functional molecule--Protein)
 20

IN SILICO DRUG DESIGN—

In silico is a term that means “computer aided. in silico drug design means rational design by which
drugs are designed/discovered by using computational methods.

According to Kubinyi , most of the drugs in the past were discovered by coincidence or trial and error
method, or in other words, serendipity played an important role in finding new drugs.

Both experimental and computational methods play significant roles in the drug discovery and
development and most of the times run complementing each other.

The main aim of computer aided drug design (CADD) is to bring the best chemical entities to
experimental testing by reducing costs and late stage attrition.

CADD involves—

a. Computer based methods to make more efficient drug discovery and development process.

b. Building up chemical and biological information databases about ligands and targets/proteins to
identify and optimize novel drugs.

c. Devising in silico filters to calculate drug likeness or pharmacokinetic properties for the chemical
compounds prior to screening to enable early detection of the compounds which are more likely to fail
in clinical stages and further to enhance detection of promising entities .

OVERVIEW OF THE PROCESS— in-silico drug design

The process of in silico drug design is an iterative one (see Fig. 1) and often proceeds through multiple
cycles before an optimized lead goes into clinical assay. The first cycle includes the cloning, purification
and structure determination of the target protein or nucleic acid by one of three principal methods: X-
ray crystallography, nuclear magnetic resonance (NMR), or homology modeling. Using computer
algorithms, compounds or fragments of compounds from a database are positioned into a selected
region of the structure (docking). These compounds are scored and ranked based on their steric and
electrostatic interactions with the target site, and the best compounds are tested with biochemical
assays. In the second cycle, structure determination of the target in complex with a promising lead from
the first cycle, one with at least micromolar inhibition in vitro, reveals sites on the compound that can be
optimized to increase potency. Additional cycles include synthesis of the optimized lead, structure
determination of the new target: lead complex| Page optimization of the lead compound. After several
cycles of the drug design process, the optimized compounds usually show marked improvement in
binding and, often, specificity for the target
 21

STRATEGIES OF IN SILICO DESIGN—

Structure Based Drug Design Ligand Based Drug Design

 22

Structure Based Drug Design—Know receptor, not know ligand

Structure based drug design (SBDD) is one of the earliest techniques used in drug design.

Drug targets are typically key molecules involved in a specific metabolic or cell signaling pathway that is
known, or believed, to be related to a particular disease state.

Drug targets are most often proteins and enzymes in these pathways. Drug compounds are designed to
inhibit, restore or otherwise modify the structure and behavior of disease related proteins and enzymes.

SBDD uses the known 3D geometrical shape or structure of proteins to assist in the development of new
drug compounds.

The 3D structure of protein targets is most often derived from X-ray crystallography or NMR techniques.
X-ray and NMR methods can resolve the structure of proteins to a resolution of a few angstroms.

However structure based drug design is not a single tool or technique. It is a process that incorporates
both experimental and computational techniques. This is generally the preferred method of drug design,
since it has the highest success rate. In the drug design stage of SBDD, docking is the preferred tool for
giving a computational prediction of compound activity.

The following steps are mostly used in SBDD—

1. Target Determination—

Drug Target: is a biomolecule which is involved in signaling or metabolic pathways that are specific to a
disease process. Biomolecules play critical roles in disease progression by communicating through either
protein–protein interactions or protein–nucleic acid interactions leading to the amplification of signaling
events and/or alteration of metabolic processes.

In structure based drug design, a known 3D structure of the target is the initial step in target
identification. This is usually determined either by X-ray crystallography or by NMR to identify its binding
site, the so called active site.

Homology Modelling : if crystallographic coordinates or a 2D NMR models are not available, then a
homology model is usually the next best way for determining the protein structure.

A homology model is a three-dimensional protein structure that is built up from fragments of

crystallographic models. Thus, the shape of an α-helix may be taken from one crystal structure, the
shape of a β-sheet taken from another structure, and loops taken from other structures. These pieces
are put together and optimized to give a structure for the complete protein.

Bioinformatics software tools are used to generate the 3D structure of the target on the basis of the
known 3D structures of templates.12 The Modeller is a popular tool in homology modeling, and
SWISS-model repository is a database of protein structures created with homology modeling .
 23

Protein Folding : Another method for target identification is protein folding. This is a difficult process
which starts with the primary sequence only .

It used to compute the confirmers and correct shape of protein. sometimes protein folding gives an
accurate model, and sometimes it gives a rather poor model. The real problem with protein folding is
that there is no reliable way to tell whether it has given an accurate model. For example, one can check
if hydrophilic residues are on the exterior of the protein and hydrophobic residues.

2. Molecular Docking— (Interaction networks)

Docking is an automated computer algorithm that determines how a compound will bind in the active
site of a protein. This includes determining the orientation of the compound, its conformational
geometry, and the scoring.

The scoring may be a binding energy, free energy, or a qualitative numerical measure.

Molecular docking is used to recognize and optimize drug candidates by examined and modeling
molecular interactions between ligand and target macromolecules. Molecular docking are used to
generate multiple ligand conformations and orientations and the most appropriate ones are
selected.15,16 There are several molecular docking tools available that includes ArgusDock, DOCK,
FRED, eHITS, AutoDock and FTDoc.

3. Scoring function—

the aim of scoring is to quantify the free energy associated with protein and ligand in the formation of
the protein-ligand interactions. Most of the docking softwares are equipped with scoring functions,
which enable computing free energy associated with protein-ligand interactions.

Wide ranges of scoring functions are available to calculate the binding between the protein and virtual
ligand.

These methods range from estimating binding by a simple shape and electrostatic complementarities to
the estimation of free energy of protein and ligand complex in aqueous solutions. Only few of them are
capable of addressing the thermodynamic process involved in the binding process.

Ligand Based Drug Design— (Don’t know receptor and know ligand)
In the past century many drug’s target proteins were unknown. The success of the design is greater if
the target is known and a structure-based drug design process can be followed. However, there are
times when there is a good reason for using a drug design without a known target.

For example, cell surface receptors make excellent drug targets, but are very difficult to crystallize. So if
homology modelling was unreliable or low identity score for the homolog protein was observed, in this
case the techniques used for structure-based drug design cannot be used. Pharmacophore models and
3D-QSAR models can be used instead. A 3D-QSAR is a computational procedure used for quantitatively
 24

predicting the interaction between a molecule and the active site of a specific target. The great
advantage of a 3D-QSAR is that it is not necessary to know what the active site looks like. Thus, it is
possible to use this technique when the target is unknown. A 3D-QSAR is a mathematical attempt to
define the properties of the active site without knowing its structure.

Assay development for hit identification—

Hit Compound— A hit compound is a molecule that shows the desired type of activity in a
screening assay. This activity should be confirmed upon retesting. The main motive is to identify
molecules that interact with the drug target.

Lead Compound— A new chemical entity that has pharmacological or biological activity and
may initiate the development of new clinically relevant compound.

Lead compounds are used as starting point in drug discovery.

Hit Identification—  Also called ‘hit-calling’. Hit identification is the most critical step to identify
compounds. This compound has ability to interact with the fully validated target.

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.

 If the structure of the

natural substrate is
known, a ligand based
approach will be
accomplished.
 25

Hit Identification Assay Feature—

 Hit and lead compounds and focused libraries can be screened against protein target within
physiological environment.
  Protein target can be membrane bound, difficult to produce for standard biochemical assays,
and difficult to purify.
 Compound cell permeability, specificity and cytotoxicity are assessed in one process .
 Process can be up scaled to screen hundreds of compounds in a few hours.

Assay Development—

First step In the drug development and toxicity testing. It is a test systems (assays) which is used to
evaluate the chemical compounds on the cellular, molecular or biochemical process.

In the recombinant era (period or age, time) majority assay used in the industry like cell based
assay(mammalian cell culture).

Creation of mammalian cell culture helps to find out the target of interest such as membrane
receptor,nuclear receptor,ion channels etc.

A "Biochemical Assay" is an analytical procedure to detect and quantify cellular processes (e.g.
apoptosis, cell signaling) or metabolic reactions. Biochemical assays are a reliable, routinely used
procedure that helps in characterizing targets and understanding of biomolecular functions.

 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.

Ex- Compound screening assays at GPCRs have have been configured (arranged or construct) to measure
the binding the affinity of a radio labeled or fluorescently labeled ligand to receptor.

To measure the compound mediated changes in of a number of second messanger metabolites

including calcium, cAMP or inositol phosphate.

Factors which is required for assay format—

A. What molecule & parameters are to be measured:

First of all should be clear that what molecule and what property of that molecule is to be
measured.

Ex— Measure the total amount of a particular protein in a cell lysate or only the phosphorylated
form or both total amount of phosphorylated protein.
 26

In the case of a protein, whether the key parameter to measure is the amount of the protein present or
its biological function, such as enzymatic activity or the effect of a cytokine on potential cellular targets.

B. Source of the molecule :

It is the important factor helps to determine the sample quality and availability.

It will also determine the concentration of the molecule and may influence its stability.

C. Quantitative Vs Semi-quantitative assay are to be used :

We should have decide that quantitative or semi-quantitative measurement of the molecule is

helpful or not Or fit for purpose.

Semi-quantitative examinations are similar to qualitative examinations; testing does

not measure the precise quantity of a substance.

We have to decide that whether (कि क्या,या, दोनों में से कौन सा) a semi-quantitative
measurement of the molecule such as western blot fulfill the requirement of the project or
whether a quantitative assay is required.

D. Number samples to be assayed :

 It is also important to consider how many samples will need to be assayed. If only a handful(
मुट्ठी भर, अल्पसंख्या) of samples will be assayed then a labour- intensive(exhaustive,
ज़्यादा), multi-step manual assay format may be perfectly acceptable .

Conversly (इसके विपरीत) if thousands or ten thousands of assays are to be run, perhaps(maybe,
probably) as part of a compound profiling exercise, it will be important to simplify, streamline
and automate the assay process as much as lab resources allow.

Assay Parameter—

A) Specificity :

It is an absolutely point which is to be considered.

We have to decided exactly what molecule and parameter of that molecule is to be

measured.

B) Sensitivity :
The important point is that the assay must be sufficiently sensitive such that the level of
the molecule falls well within the dynamic range of the assay.
 27

If great sensitivity is required then a fluorescence, rather than absorbance, the readout is
more likely to give the desired sensitivity. Sensitivity may also be increased by utilising
enzymatic amplification of the original signal.

C) Dynamic range :
It is important to determine the dynamic range of the assay. In other words the range
over which the assay readout is proportional to the amount of target molecule in the
sample being analysed. In the case of an assay to measure the concentration of a
molecule, it is important that the assay (regardless of the format adopted) is
appropriately calibrated by the construction of a suitable calibration curve such as the
hypothetical example.

D) Reproducibility/robustness/accuracy :

The data is obtained from the assay should be robust, reproducible and reliable(authentic).

The assay should be robust and does not affected by changes in the sample preparation &
handling.

Equally, it should give the same result regardless of the individual operating assay.

The assay should be highly reproducible (also referred to as precision) such that the
degree of variation is as small as possible both on an intra and inter assay basis.

If the assay is to measure the absolute (rather than relative) amount of a molecule then it
must be calibrated against an accepted standard in order to give accurate quantitation.
Sometimes the assay workflow requires significant sample workup (e.g., concentration,
removal of interfering sample components etc..).

The levels of robustness, reproducibility and accuracy that are acceptable will depend on
the purpose of the assay and must be decided upon by the researcher for their own
particular current use.

Hit Identification Assay Applications—

 Primary and secondary medium to high throughput screening in drug discovery    

 Target identification or validation for hit phenotypic screening

Protein Structure—
Egg whites contain large amounts of proteins called albumins, and the albumins normally have a specific
3D shape, thanks to bonds formed between different amino acids in the protein. Heating causes these
 28

bonds to break and exposes hydrophobic (water-hating) amino acids usually kept on the inside of the
protein.

A) Primary structure:

The simplest level of protein structure, primary structure, is simply the sequence of amino acids in a
polypeptide chain. For example, the hormone insulin has two polypeptide chains.

The sequence of a protein is determined by the DNA of the gene that encodes the protein (or that
encodes a portion of the protein, for multi-subunit proteins). A change in the gene's DNA sequence may
lead to a change in the amino acid sequence of the protein. Even changing just one amino acid in a
protein’s sequence can affect the protein’s overall structure and function.

Protein structure is hierarchical

Importance of primary structure

 To predict secondary & tertiary structures from sequence homologies with related proteins à
structure prediction.

 Many genetic diseases result from abnormal amino acid sequences.

 To understand the molecular mechanism of action of proteins.

 To trace evolutionary paths.

 29

Methods of amino acid sequence determination

 End group analysis – Edman degradation

 Gene sequencing method

Secondary Structure:

• The polypeptide chain of a protein is folded into a specific 3-D structure producing a protein’s
unique conformation.

• It consists of α - helix

β – pleated sheet

β - bends

Non - repetitive structures

Super secondary structures

 The secondary structure is determined by the chemical interactions (mainly hydrogen bonding)
of amino acid residues with other amino acids in close proximity.

• Two types of β pleated sheet or β structure commonly occur in proteins :-

Anti-parallel sheets may be formed by extended adjacent segments of polypeptide chain, whose
sequences with respect to the direction from –NH 3+ to –COO- run in opposite direction.

Chains running in the same direction form the parallel pleated sheet.

• Along a single β strand, the amino acid side chains are positioned alternately up and down.

• The polypeptide chains in many proteins often fold sharply back upon themselves, giving rise to
secondary structures called β bends, which are stabilized by H-bonds.

• Proteins are generally build up from combinations of secondary structure elements, α-helices &
β-sheets. Secondary structural elements combine in ways that result in formation of a stable
hydrophobic core.
 30

Motifs & Domains—

Domains—

A structural domain  is an element of the protein's overall structure that is stable and
often folds  independently of the rest of the protein chain.e.g., the calcium-binding
domain  of calmodulin.

Secondary structural elements & motifs combine to form domains

Motifs—

Protein motifs  are small regions of protein three-dimensional structure or amino acid sequence shared
among different proteins. They are recognizable regions of protein structure that may (or may not) be
defined by a unique chemical or biological function.

Tertiary Structure :

The overall three-dimensional structure of a polypeptide is called its tertiary structure. The tertiary
structure is primarily due to interactions between the R groups of the amino acids that make up the
protein.

This 3-D organization revealed by à Crystallography & Nuclear magnetic resonance spectroscopy.

This level of organization is determined by the non-covalent interactions between helices & β-structures
together with the side chain & backbone interactions unique to a given protein.

Quaternary structure :

Many proteins are made up of a single polypeptide chain and have only three levels of structure.

However, some proteins are made up of multiple polypeptide chains, also known as subunits. When
these subunits come together, they give the protein its quaternary structure.

Schematic illustration of isologous and

heterologous association between
protein subunits. (A) Isologous
association to form a dimer. (B)
Heterologous association leading to an
infinitely long polymer. (C) Heterologous
association to form a closed, finite
structure, in this case a tetramer.
31
 32

Computational Prediction of protein structure—

Most modern drug discovery projects start with protein target identification and verification to obtain a
verified drug target. For structure-based drug design the three-dimensional structure of the protein
needs to be determined experimentally by using either x-ray crystallography or nuclear magnetic
resonance (NMR) spectroscopy [1]. While both methods are increasingly being applied in a hight
hroughput manner, structure determination is not yet a straightforward process. X-ray crystallography is
limited by the difficulty of getting some proteins to form crystals, and NMR can only be applied to
relatively small protein molecules.

 Various computational tools have been developed that predict different levels of protein structural
hierarchy.

PROCLAS Prediction of structural class of proteins such as Alpha or Beta or Alpha+Beta or Alpha/Beta
S

PSA This server allow user to analysis of protein sequence and present the analysis in Graphical and Textual format.
property plots of 36 parameter (like Hydrophobicity Plot, Polarity, Charge) of single sequence

and multiple sequence alignment 

CHpredict The CHpredict server predict two types of interactions: C-H...O and C-H...PI interactions.

Homology Modeling Methods—

The term "homology modeling", also called comparative modeling or template-based modeling (TBM),
refers to modeling a protein 3D structure using a known experimental structure of a homologous
protein (the template).

 Structural information is always helps to study of protein function, dynamics, interactions with ligands
and other proteins.

The "low-resolution" structure provided by homology modeling contains sufficient information about
the spatial arrangement of important residues and may guide the design of new experiments.

In some cases modeling is combined with electron microscopy or small-angle X-ray scattering (SAXS)
data to generate low-resolution models of a protein complex .
 33

the method of homology modeling usually provides the most reliable results. The use of this method is
based on the observation that two proteins belonging to the same family (and sharing similar amino acid
sequences), will have similar three-dimensional structures. This is possible because the degree of
conservation of protein three-dimensional structure within a family is much higher than the
conservation of the amino acid sequence.

General steps for making a homology model:

 identification of a template - related protein with a known experimental

structure of good resolution;
 amino acid sequence alignment;
 alignment correction;
 backbone generation;
 generation of loops;
 side chain generation & optimization;
 ab initio loop building in regions in which template does not have
structure;
 overall model optimization;
 model verification using quality criteria
See the review article of proteomics

Application of NMR

Protein structure prediction by NMR

 34

• Protein production

• Protein purity

• Isotopic labeling

• NMR samples / conditions / tubes

• Simple spectra / evaluation (stability, tertiary structure)

Protein production :

• protein sample(s)

• in theory, as little as a few mg of protein is sufficient

-best if the protein sample for NMR is > 1 mM

• in practice, tens of milligrams (or more) are usually necessary,

as are multiple samples

-multiple samples if protein is not stable

-multiple samples with different isotopic labeling schemes

• many bacterial expression vectors/cell strains are available for expression in bacteria. -good track
record, easily automated

• expression in eukaryotic cells more complicated (yeast, insect, human cells)

 35

Protein purity :

• protein samples should always be as pure as possible

• in practice, for small proteins, small amounts of high molecular weight contaminants are OK

Isotopic labeling of protein for NMR

• protein must be uniformly isotopically labeled with 13C and 15N

bacterial expression

cell strains grow well on minimal media (D-glucose (U-13C6, 98-

99%) and 15NH4Cl (98-99%) as sole carbon and nitrogen sources,
respectively)

• There are also alternatives to minimal media (i.e. isotopically

labeled rich media), but they are much more expensive

The NMR sample:

• buffer: no C- or N-bound protons

• phosphate is the best….

• deuterated Tris, deuterated acetate, deuterated imidazole, etc., are OK (can be expensive)

• salts K+, Na+, Cl-, SO4

2-, etc., all OK (no protons)

• too much salt leads to decreased S/N

 36

• pH: neutral or lower is best

• must minimize the rate of exchange of amide protons with solvent

• solvent • 90% H2O, 10% D2O 90% H2O, 10% D2O (for instrumental lock.

• temperature

• sample dependent: usually 25 to 35 °C

• bacteriostatic agents

• sodium azide used widely

• put it all together in a good quality, clean NMR tube

• “standard” NMR tube is 5 mm diameter

(for use in a 5 mm NMR probe)….volume

of sample is ~500 - 700 uL (~1 mM protein)

• magnetic susceptibility matched tubes,

“Shigemi” tubes, permit lower volume samples to be used (i.e. less sample, or more concentrated
sample), usually without deleterious effects (but the tubes are

complicated)…volume of sample is 200-300 uL

37
 38

Rational Drug Design—

Rational drug design refers to the development of medications based on the study of the structures
and functions of target molecules.

During rational drug design, researchers take three general steps to create a new drug:
Step 1. Identify a receptor or enzyme that is relevant to a disease they are going to design a drug
for.
Step 2. Elucidate the structure and function of this receptor or enzyme.
Step 3. Use the information from step two in order to design a drug molecule that interacts with the
receptor or enzyme in a therapeutically beneficial way.

Traditional drug design :

Traditional drug discovery (also referred to as forward pharmacology) is a trial-and-error based method
of drug discovery, in which a chemical compound is tested with cultured cells or animals, and then
applied to humans.
 39

Traditional drug discovery involves the origin of drug discovery that evolved in natural sources,
accidental events.

It was not target based and not much systemized as today.

Methods for traditional drug design :

A) Random screening-

it includes random screening of synthetic compounds or chemicals or on natural products by bioassay

procedures. Involves two approaches:

1.Screening for selected class of compounds like alkaloids, flavonoids, etc

2. Screening of randomly selected plants for selected bioassays

B) Trial and error method-

Trial and error method includes berries, roots, leaves and barks could be used for medicinal purposes to
alleviate symptoms of illness.

Examples :Willow bark –contains salicin –fever reducing in general

• Cinchona bark – contains quinine – fever associated with malaria

• Chinese herbal remedies – used to treat many illness.

C) Ethnopharmacology approach-

Ethnopharmacology aims to identify new therapeutic agents based on their traditional use.

It begins by the identification of disease states, and of the traditional therapies for these, most
commonly herbals.

Herbals of interest are selected from ethnopharmacological surveys, and tested on experimental
models of the diseases of interest.

Andrographis paniculata was used for dysentery in ethnomedicine and the compounds responsible for
the activity were isolated as andrographolide.

•Morphine from Papaver somniferum,

Contributions of Ethnopharmacology

Discovery of artemisinin from Artemesia alba for malaria,

•guggul sterones from Commiphora mukul (for hyperlipidemia), boswellic acids from Boswellia serrata
(antiinflammatory).
 40

• bacosides from Bacopa monnieri (nootropic and memory enhancement) was based on the leads from
these codified systems of medicine prevailing in China and India.

D) Serendipity method-

Serendipity” refers to “an accidental discovery;” i.e, “finding one thing while looking for something else.

No scientific discovery has ever been made by pure luck.

The serendipitous discovery of penicillin in 1928 by Alexander Fleming occurs.

Fleming was engaged in research on influenza when one of his staphylococcus culture

plates had become contaminated and developed a, mold that created a, bacteria-free circle

•Fleming recognized the possible significance of the bacteria-free circle and by isolating the mold in pure
culture Serendipity method.

E) Classical pharmacology-

Also known as function based approach.

Anciently, drug discovery programmes were often based-successfully-on measuring a complex response
in vivo.

•Such as prevention of experimentally induced seizures, lowering of blood sugar, or suppression of an

inflammatory response.

•Without the prior identification of a drug target.

Rational Drug design-

Structure Based Drug Design "_ _#