Drug Discovery using

Biotechnology
Gopal Agarwal
Ph.D Research Scholar
NIPER-Ahmedabad
Importance of Biotechnology in Drug
discovery
• Biotechnology has produced more than 200 new therapies
and vaccines, including products to treat cancer, diabetes,
HIV/AIDS, and autoimmune disorders
• more than 400 biotech drug products and vaccines currently
in clinical trials, targeting more than 200 diseases, including
various cancers, Alzheimer’ s disease, heart disease,
diabetes, multiple sclerosis, AIDS, and arthritis
• An average approval of 10 – 15 products a year indicates
that pharmaceutical biotechnology is a highly active sector
• Biotechnological companies are said to be more
entrepreneurial, nimble and fast
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 India Biotech Market
• India is ranked 12th in world in Biotech market and 3rd in
Asia Pacific
 USD 3.7 Billion to be spent on biotechnology from 2012-
17
•No.1 producer of Hepatitis B vaccine recombinant
•USD 100 Billion industry by 2025

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World top 5 most expensive medicine
• Glybera ( not approved in US but approved in Europe
market)-gene therapy for lipoprotein lipase deficiency-
$1.21 million/year
• Soliris- PNH (paroxysmal nocturnal haemoglobinuria)-
$440k/year
• Vimizim- enzyme replacement therapy for Morquio A
syndrome- $380k/year
• Elaprase- Hunter Syndrome- $375k/year
• Naglazyme- Maroteaux-Lamy Syndrome- $365k/year

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 Know the cause of disease
Cell cultures, Cross Species studies, Bioinformatics, Biomarker, Proteomics

Target Selection
8000 therapeutics targets( inhibitors, modulators and enhancers)

Drug selection
Based on target and disease pathology

Drug Development

Clinical Trials
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Application of Biotechnology in
Drug Discovery
• Two main application
Development of traditional small molecule drug
Allows researchers to focus drug discovery on a single
molecular target
Techniques includes Molecular Biology, Biochemical and
Biophysical methods
Target identification, Target validation, Target Protein
expression
Generation of Drug screening assay

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Contd…
Development of Protein Therapeutics
 traditional small molecules approach + Recombinant
Technology in the synthesis or expression of Protein
therapeutics
E.g.: Antibodies

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 Technology for target Validation
• Transgenic mice
• Knock out mice
• Impact of gene deficiency on induction of the disease in
Knock out mice gives important clues on the causative role of
the target
• Conditional Knock out : Disease induction in the adult animal
with the gene in the “on” state and gene is turned off by a
generic switch
• siRNA (small interfering ribonucleic acid) technology

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 Role of Biotechnology in Drug
targeting
• Targeting using a specific vector molecules
• Vector molecules have affinity towards ligand
characteristics for a target tissue
• Antibodies; peptides; lectines and hormones

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Drug delivery
• Immunotoxin

A
Fusion of Immunotoxin
an
Splitting antibody

• A: active subunit (toxin) ; B: cell binding subunit

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 Other Pathway
• Inverted micelle formation
• Carpet model
• Pore formation model
• Macro-pinocytosis
• Endocytosis
• Direct translocation

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Journal of Pharmaceutical Investigation;2016
High throughout screening
• Cell based assay
• In vitro biochemical assay
• Cell based assay: three broad categories
 Second messenger assay
Reporter gene assay
Cell proliferation assay
Yan et al.: G protein coupled receptor activation using β
galactoside enzyme complementation technology
Flow cytometry : Waller et al. used flow cytometry as a
technique for studying GPCR assembly, pharmacology and
screening

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Detection technologies for HTS
screening
• FRET (Fluorescence resonance energy transfer)
• FP (Fluorescence polarization)
• TRF(time resolved Fluorescence)
• FLM(Fluorescence lifetime measurements)
• FCS (Fluorescence correlation spectroscopy)
• FPR (Fluorescence photobleaching recovery)
• MB ( Molecular beacons)

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Application of Fluorescent techniques
• To identify protein interaction
• Monitor intercellular signalling activities in real time
• Survey Bioactive molecules
• Dissociation constant can be determined (Kd)
• Association constant
• Competitive binding assay

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3D cell cultures in Drug Discovery
• Provides more accurate microenvironment where cell resides
in tissue
• 3D cells cultures grow cells into 3D aggregates or spheroids
using a scaffold matrix or scaffold free matrix
• Most common used scaffold- BD Matrigel basement
membrane matrix (BD Sciences), Cultrex (basement
membrane extract) (BME; Trevigen), and hyaluronic acid are
commonly used biologically derived matrixes
• Polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polylactide-co-glycolide (PLG), and polycaprolactone (PLA)
are common materials used to form synthetic scaffolds
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3D cell cultures as Biosensors
• Simple, fast, and cost-effective alternative to large-scale and
cost-intensive animal testing
• Cells grown in 3D culture can be incorporated into a biosensor
either by direct attachment onto a biotic or abiotic substrate
surface or by indirect attachment via entrapment in a
biocompatible biopolymer
• Substrate selection depends on both the cell line and the
sensor’s application; most biosensors use silicon, glass, or plastic
surfaces
• Most commonly used transduction techniques for 3D cell-based
biosensors is the electrical/electrochemical biosensor

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Bioinformatics

• Pymol and Rasmol: Best fit of ligand

• Gene logic : for understanding underlying mechanisms of
diseases, discovery and prioritization of gene targets &
biomarkers
• Clindex: e-clinical trial

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Protein Engineering in Drug Discovery
• Improves the pharmco-dynamics profile to obtain a drug
that acts faster or slower
• Development of pharmacological half life and development
of controlled released kinetics
• Alteration of receptor binding specificity
• Reducing the immunogenicity of the protein

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Mutation in drug discovery
• Improve the pharmacokinetic and pharmacodynamic activity,
• develop antagonist
• E.g. Insulin Lispro (May 1996) : Switched B28 and B29 of proline and
lysine reduced the association affinity by 300 - fold, resulting in faster
uptake and action, as well as shorter half - life
• Tissue plasminogen activation factor (Reteplase): a deletion mutant
was constructed to reduce binding of the protein at hepatocytes via
the EGF - domain( epidermal growth factor ) that increased half -
lives of 13 – 16 min and fivefold activity

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Post Translation Engineering
• Post – translational biosynthesis today is the covalent
attachment of a chemical group, not a mandatory
glycosylation, but attaching fatty acids or PEG - chains
alteration of a pre – existing post - translational
modification
• Mostly carried out in CHO (Chinese Hamster Ovary) and
Baby Hamster Kidney cell lines
• Plant system (such as carrot cells) ; Saccharomyces
cerevisiae and Pichia pastoris
• PEGylation : alters the physical, chemical and biological
profile of a protein
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Natural Products production
Biotechnologically
Use of mostly actinomycetes as a strain
for natural product synthesis

Use of E.coli
for the production of 6 MSA and
erythromycin
For isoprenoid synthesis

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Biomarkers in Drug Discovery
• Critical component of a modern drug discovery project
• Helps to know the severity and progressive pathway of a
disease
• Bridge between animal models and patient
• Helps to assess the impact of the development candidate on
the intended target
• Change in biomarker in response to the therapeutic target
• When the biomarker indicates complete target inhibition
but the disease still does not respond it is likely that the
target is inappropriate for the disease

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Drug Discovered using Biotechnology

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 Conclusion
• Biotechnology plays a crucial role in drug discovery
• Its has a significant application in drug targeting, target
validation
• In HTS , Biotechnology provides a sensitive and flexible
method of detection
• Biotechnology covers a major area of world pharmaceutical
companies
• Biotech drug covers a large sector of pharmaceutical market

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References
• Drug Discovery and Evaluation:Pharmacological assays; H.
Vogel; Springer; 3rd edition; Volume 1; (2008) 9-35.
• Pharmaceutical Biotechnology,Drug Discovery and Clinical
Applications; O. Kayser , H. Warzecha; Wiley-Backwell;2nd
edition; Volume 1; (2012) 5-50.
• Development of FRET Assay into Quantitative and High-
throughput Screening Technology Platforms for Protein–
Protein Interactions ; Y. Song, V. Madahar,J. Liao ;Annals of
Biomedical Engineering, 39, (2010) 1224–1234.

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References
• Drug targeting; V .Torchilin; European Journal of Pharmaceutical
Sciences; 11(2000) S81–S91.
• Cell penetrating peptides as an innovative approach for drug delivery,
then, present and the future; S.Bashyal et al.; Journal of
Pharmaceutical Investigation; 6 (2016) 25-35.
• Biotechnology and genetic engineering in the new drug development,
Part I, DNA technology and recombinant proteins; A. Stryjewska et
al., Pharmacological Reports; 65 (2013) 1075-1085.

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