Drug screening methods by
using chemical, biological and
analytical tests
Dr Parthajyoti Neog, SRD
Department of Pharmacology
Contents
1. Introduction
2. Drug discovery cycle
3. Methods of drug screening
4. Newer tools of drug screening
5. Challenges of drug screening
6. Conclusion
Introduction
Drug screening is the process by which
potential drugs are identified and optimized before
selection of a candidate drug to progress to clinical
trials.
It can involve screening large libraries of chemicals for a
particular biological activity in high-
throughput screening assays.
Introduction cont..
Pharmacological research started in Europe in the
second half of the 19th century when their founders,
e.g., Rudolf Buchheim and Oswald Schmiedeberg,
investigated the action of existing drugs in animal
experiments.
Father of Pharmacology Founder of modern Pharmacology
Rudolf Buchheim Oswald Schmiedeberg
Introduction cont..
The classical way of pharmacological screening involves
sequential testing of new chemical entities or extracts
from biological material in isolated organs followed by
tests in whole animals, mostly rats and mice but also
higher animals if indicated.
Most drugs in use nowadays in therapy have been found
and evaluated with these methods.
Methods of drug screening
1. In vitro:
Experimental process in a given procedure which is
mainly done outside the body in a controlled condition.
Activity assays (screen the activity)
Bioassays (define the molecular mechanism)
Toxicity assays (Toxicity of chemicals)
Types:
Biological assay using isolated tissues/organs.
(skeletal/smooth muscles, aorta, heart etc.,)
Chemical Assay using regents-
Antioxidant assays
Xanthine oxidase activity
Antiglycation activity
DNA, protein, RNA level assays
Immunological assays
Cell culture studies
Toxicity(cyto) assays
Immunological assays
Cancer cell line studies
2.Ex vivo: Experimental process which is performed
outside the living body in an ‘artificial in vivo
environment’ This usually lasting up to 24 hrs
3.In vivo: Experimental process which is performed
in the living body using laboratory animals
4.In silico: Process which is performed on computer
or via computer simulator
Bioassay (Biological screening)
An assay is an analytical measurement procedure
defined by a set of reagents that produces a
detectable signal for quantifying a biological
process.
Bioassay is the determination of the potency of
chemical and biological agents like drugs, hormones,
ions, etc.,
Uses whole animals, isolated organs and tissues or
using cell lines (biological indicators).
Biological Indicators:
Body temperature
Blood glucose level
Behavioral responses
Serum parameters
Contraction/relaxation
Growth/inhibition of cells
Animal studies
STEP I
Toxicological assessment of chemicals
LD50 estimation
Using Acute/sub-acute, chronic etc., studies
STEP II
Evaluation of Pharmacological activity
Animal models of induced disease and injury
STEP III
ADME Studies
Absorption studies
Tissue/organ/fluid conc. estimation
Histopathological studies
Serum estimation of biological indicators for drug &
metabolites Factors:
Species
Strains
Sex
Age
Disease
Induction
Environmental
Antioxidant assays
• There are two general types of assays widely used for
different antioxidant studies.
1. Lipid peroxidations, including-
-Thiobarbituric acid assay (TBA),
-Malonaldehyde/high-performance liquid
chromatography (MA/HPLC) assay,
-Malonaldehyde/gas chromatography (MA/GC)
assay,
-β-carotene bleaching assay, and
-Conjugated diene assay.
2. Associated with electron or radical scavenging,
including-
-2,2-diphenyl-1-picrylhydrazyl (DPPH) assay,
-Ferric reducing/antioxidant power (FRAP) assay,
-Ferrous oxidation−xylenol orange (FOX) assay,
-Ferric thiocyanate (FTC) assay, and
-Aldehyde/carboxylic acid (ACA) assay.
Xanthine oxidase activity
• Xanthine oxidase is a form of xanthine
oxidoreductase, a type of enzyme that
generates reactive oxygen species.
Antiglycation activity
• Glycation is a non enzymatic condensation reaction
between reducing sugars and amino groups of proteins
that undergo rearrangements to stable ketoamines,
leading to the formation of advanced glycation end
products (AGEs).
• Increased glycation and build-up of tissue AGEs have
been implicated in diabetic complications because they
can alter enzymatic activity, decrease ligand binding,
modify protein half-life and alter immunogenicity
Newer tools for drug screening
Combinatorial chemistry
• Comprises chemical synthetic methods that make it
possible to prepare a large number (tens to
thousands or even millions) of compounds in a
single process.
• These compound libraries can be made as
mixtures, sets of individual compounds or chemical
structures generated by computer software.
• Combinatorial chemistry can be used for the
synthesis of small molecules and for peptides.
Cassette dosing
Microassay
• DNA recombinant biotechnology
• Sensitive ELISA
• Reverse transcriptional PCR
• Riboneuclease protection assays
• cDNA microarrays
Biochemical assays
• Enzyme assays
• Protein-protein interaction
• Membrane receptor-ligand binding assays
• Soluble receptor-ligand binding assays
• Microdialysis
Microbe-based screening assays
Modern analytical methods
1. Atomic absorption spectroscopy
2. Counter counting
3. Fluorescence activated cell sorting (FACS)
4. Positron emission scanning
5. MRI and Spectroscopy
1.Atomic absorption spectroscopy
• Estimates the conc. of various metal ions of
physiological significance, such as Na, K, Ca, Fe, Cu, Zn
from biological fluid or tissue extracts.
• Tissue extracts are prepared in 0.3N perchloric acid by
sonication at low wattage and micro centrifuged at
14000 rpm. Supernatants are used in atomic absorption
spectroscopy.
Fig- Representative diagram of AAS
2. Coulter counting
• A Coulter counter is an apparatus for counting and
sizing particles suspended in electrolytes.
• It is used for cells, bacteria, prokaryotic
cells and virus particles.
• The Coulter principle states that particles pulled through
an orifice, concurrent with an electric current, produce
a change in impedance that is proportional to the
volume of the particle traversing the orifice.
3.Fluorescence activated cell
sorting (FACS)
• Fluorescence-activated cell sorting (FACS) is a technique
to purify specific cell populations based on phenotypes
detected by flow cytometry.
• This method enables researchers to better understand
the characteristics of a single cell population without
the influence of other cells
4. Positron emission scanning
5. MRI and Spectroscopy
Challenges in drug screening
• Drug development is a lengthy, complex, and costly
process, entrenched with a high degree of
uncertainty that a drug will actually succeed.
• The unknown pathophysiology for many nervous
system disorders makes target identification
challenging.
• Animal models often cannot recapitulate an entire
disorder or disease.
• Challenges related to heterogeneity of the patient
population might be alleviated with increased clinical
phenotyping and endotyping.
Challenges in drug screening cont..
• Greater emphasis on human data might lead to
improved target identification and validation.
• There is a lack of validated diagnostic and
therapeutic biomarkers to objectively detect and
measure biological states.
• Unfamiliarity with current regulatory processes for
investigational new drug (IND) applications can be
resolved through pre-IND meetings.
Conclusion
• High throughput screening is one of the most
prominent domains in terms of drug discovery
which has a pivotal contribution for a new
molecule to turn in to a therapeutic entity.
• Emergence of a new diseases and failing
treatment of existing diseases has increased the
importance of HTS in a greater extent.
Conclusion cont..
• The combinatorial approach of the drug development
scientists and engineering has made the never
approaching dream a reality
• This amalgamation of technology of every field is
expected to increase to multiple folds in the next
decade and would be creating a method through which
more and more safe drug would be discovered for all
the diseases.
Reference
1. Drug Discovery and Evaluation: Pharmacological
Assays Editors: Vogel, Hans Gerhard (Ed.)
2. Drug Screening Methods: Preclinical Evaluation
of New Drugs: Gupta SK
