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Drug Discovery using Biotechnology

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 disordersmore 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 arthritisAn average approval of 10 15 products a year indicates that pharmaceutical biotechnology is a highly active sectorBiotechnological 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 3.7Billion to be spent on biotechnology from 2012-17No.1 producer of Hepatitis B vaccine recombinantUSD 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/yearVimizim- enzyme replacement therapy for Morquio A syndrome- $380k/yearElaprase- Hunter Syndrome- $375k/yearNaglazyme- Maroteaux-Lamy Syndrome- $365k/year

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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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ContdDevelopment 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 miceKnock 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 19-09-201610

Drug delivery Immunotoxin

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

Splitting A B Fusion of an antibody Immunotoxin 19-09-201611

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Other PathwayInverted micelle formation Carpet model Pore formation model Macro-pinocytosisEndocytosisDirect translocation

Journal of Pharmaceutical Investigation;201619-09-201613

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 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 tissue3D cells cultures grow cells into 3D aggregates or spheroids using a scaffold matrix or scaffold free matrixMost 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 BiosensorsSimple, fast, and cost-effective alternative to large-scale and cost-intensive animal testingCells 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 sensors application; most biosensors use silicon, glass, or plastic surfacesMost commonly used transduction techniques for 3D cell-based biosensors is the electrical/electrochemical biosensor19-09-201618

Bioinformatics

Pymol and Rasmol: Best fit of ligand Gene logic : for understanding underlying mechanisms of diseases, discovery and prioritization of gene targets & biomarkersClindex: 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 19-09-201620

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 - lifeTissue 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 19-09-201621

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 modificationMostly carried out in CHO (Chinese Hamster Ovary) and Baby Hamster Kidney cell lines Plant system (such as carrot cells) ; Saccharomyces cerevisiae and Pichia pastorisPEGylation : 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 projectHelps to know the severity and progressive pathway of a diseaseBridge 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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Conclusion Biotechnology plays a crucial role in drug discovery Its has a significant application in drug targeting, target validationIn 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 19-09-201627

ReferencesDrug 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 ProteinProtein Interactions ; Y. Song, V. Madahar,J. Liao ;Annals of Biomedical Engineering, 39, (2010) 12241234.19-09-201628

Drug targeting; V .Torchilin; European Journal of Pharmaceutical Sciences; 11(2000) S81S91.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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