PC-611

BIO-CHEMICAL
ASSAY

KAJAL GUPTA
PA/2019/108
PHARMACEUTICAL ANALYSIS
DRUG DEVELOPMENT PROCESS

Receptor Chemical assay Primary Chemistry Pre-clinical

Enzyme Immunoassay Secondary SAR Clinical phase 1
Hormone Bioassay ADME Clinical phase 2
Bioavailability
DNA Microbiological assay Clinical phase 3
In-vitro and in-vivo efficacy
Biochemical assay
‘

Target identification Screening Lead identification

Assay development Clinical trials
Target validation Hit to lead target Lead optimization

Hughes, J. P., Rees, S., Kalindjian, S. B. & Philpott, K. L. Principles of early drug discovery. British journal of pharmacology 162, 1239-1249 (2011).
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PARADIGMS IN DRUG DISCOVERY

Physiology- Target-
based based
Target-unknown Target-known

Read outs are based on the

Physiological/phenotypic
activity or expression of
read outs
the target

Cell based or biochemical

Cell based assay
assay

Note: the two paradigms are not mutually exclusive: A drug discovery program can use two-pronged approach

Hughes, J. P., Rees, S., Kalindjian, S. B. & Philpott, K. L. Principles of early drug discovery. British journal of pharmacology 162, 1239-1249 (2011).
Tuesday, January 25, 2022 3
TYPES OF TARGET BASED ASSAY
 Measure function of a purified target

 Identify compounds that modulate activity / binding of

the target protein
 Examples: Kinase/ATPase assays, protease assays,
 Bio-chemical assay protein interaction assays

 Cell-based assay  Measure function of the target in the context of the cell

 Measure expression of the target

 Provide a functional read-out of compound activity

 Examples: reporter assays, viability assays, GPCR and

ion channel assays, qPCR

Hughes, J. P., Rees, S., Kalindjian, S. B. & Philpott, K. L. Principles of early drug discovery. British journal of pharmacology 162, 1239-1249 (2011).
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FEATURES OF ASSAY DEVELOPMENT

 Sensitive

 Specificity

 Repeatability

 Reproducibility

 Validity

 Stability

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BIO-CHEMICAL ASSAY

 Biochemical assay is assay of substances which are found in living organisms and chemical reactions underlying the life
processes
 Types of biomolecules:

a) Lipid, proteins, polysaccharide, glycolipids, sterols

b) Vitamins

c) Hormones and neurotransmitters

d) Metabolites

e) Monomers, oligomers, polymers

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BIOCHEMICAL ANALYSIS TECHNIQUES

Chromatography

Hybridoma ELISA

Radioimmuno-
assay

Electrophoresis Spectrophotometry

Inglese, J. et al. High-throughput screening assays for the identification of chemical probes. Nature chemical biology 3, 466-479 (2007).
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ASSAY FOR PROTEIN-PROTEIN INTERACTIONS: TAU-FYN
INTERACTIONS (ALPHA SCREEN ASSAY)

• Interaction between tau and Fyn active proteins implicated in Alzheimer's

• Donor beads labelled with glutathione-binds to Fyn GST
• Acceptor bead chelated to nickel-binds to His-tag on Tau protein

Cochran, J. N. et al. AlphaScreen HTS and live-cell bioluminescence resonance energy transfer (BRET) assays for identification of tau–fyn SH3 interaction
inhibitors for Alzheimer disease. Journal of biomolecular screening 19, 1338-1349 (2014). Tuesday, January 25, 2022 8
ENZYME ASSAY: MONOAMINE OXIDASE INHIBITION ASSAY
 MAO source- Rat brain mitochondria

 Inhibitor- Selegiline ( Specific MAO-B inhibitor)

 Inhibitor- Benzylamine (Highly selective MAO-B inhibitor)

• Rat • Dissect brain • Centrifuge • Protein content in

• Anesthetize • Homogenize with • Collect crude mitochondrial pellets
• Perfuse and dissect PBS (Mitochondrial determined by
pellets) Barford protein assay
• Wash these pellets at
11000 g

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MONOAMINE OXIDASE INHIBITION ASSAY
Blank Control Standard Sample
inhibitor

PBS + Benzyl amine PBS +MAO + Substrate PBS + MAO + PBS +MAO + Sample
Substrate + Selegiline (Different conc.)+
Substrate

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IMMUNOASSAY- ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA)

 Use of antigen antibody complexes and means of generating measurable results

 Detects substances, mostly proteins with antigenic properties

 Produces color based on enzymatic reaction

 Advantage-sensitive and fast

 Disadvantage-deals with radioactivity

 Application: HIV test

Crowther, J. R. The ELISA guidebook. Vol. 149 (Springer Science & Business Media, 2001).
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TYPES OF ELISA
Direct Indirect Sandwich

Substrate

Enzyme Product
Substrate

Product
Enzyme
Substrate

Secondary antibody
Enzyme Product

Antigen
Primary antibody Primary antibody

Antigen Antigen Captured antibody

Crowther, J. R. The ELISA guidebook. Vol. 149 (Springer Science & Business Media, 2001).
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CHEMICALS USED IN ELISA
Enzyme Substrate Chromogen
• Horseradish peroxidase • H2O2 • Tetramethyl benzidine

• Alkaline phosphatase • Nitrophenyl phosphate • Diethandamine+MgCl2

CALCULATIONS
• Absorbance checked by microplate reader

Optical density
• Optical density Vs Log of concentration is plotted

• Standard curve prepared by known concentration of

Log conc.
antigen

Crowther, J. R. The ELISA guidebook. Vol. 149 (Springer Science & Business Media, 2001).
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THANK YOU

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