Drug Discovery using Biotechnology

Gopal AgarwalPh.D Research Scholar

NIPER-Ahmedabad

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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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Clinical Trials

Drug Development

Drug selection Based on target and disease pathology

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

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

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Application of Biotechnology in Drug Discovery • Two main application Development of traditional small molecule drugAllows researchers to focus drug discovery on a single

molecular targetTechniques 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: active subunit (toxin) ; B: cell binding subunit

Splitting

A

B

Fusion of an antibody

Immunotoxin

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

Journal of Pharmaceutical Investigation;2016

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High throughout screening • Cell based assay • In vitro biochemical assay • Cell based assay: three broad categories Second messenger assayReporter gene assay Cell proliferation assayYan 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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• 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.

References