Target Validation

Target Validation
Introduction,Drug discovery, Target identification and validation, Target validation and techniques
By
Ms. B. Mary Vishali
Department of Pharmacology

1. 1 TARGET VALIDATION A Seminar as a part of curricular requirement for I year M. Pharm I I semester Presented by Ms. B. Mary Vishali (Reg. No. 20L81S0104) Department of Pharmacology Under the guidance/Mentorship of Dr. P. Ramalingam., Ph.D. Director- R&D Division, Professor of Pharmaceutical Analysis and Medicinal Chemistry.

2. 2 Introduction Drug discovery Target identification and validation Target validation and techniques References CONTENTS:

3. 3 Introduction A drug discovery program initiates because there is a disease or clinical condition without suitable medical products available The aim is to achieve registration by one or more regulatory authorities, to allow the drug to be marketed legally as a medicine for human use. Developing a new drug is a complex process which can take 12–15 years and cost in excess of $1 billion.

4. 4 Drug Discovery and Development:

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6. 6 Target identification and validation One of the most important steps in developing a new drug is target identification and validation. A target is a broad term which can be applied to a range of biological entities which may include for example proteins, genes and RNA. A good target needs to be efficacious, safe, meet clinical and commercial needs and, above all, be ‘druggable’. Ex: Proton pump (H+K +ATPase) of parietal cells of stomach

7. 7 Strategies used in target discovery Two main strategies are employed; 1. Molecular approach: Target discovery through understanding of cellular mechanisms (ex: Tissues, cell lines) 2. Systems approach: Target discovery through the study of disease in whole organism (Clinical sciences, patient/animal models)

8. 8 Target validation Once a gene target or a mechanistic pathway is identified, the next step is to demonstrate that it does play a critical role in disease initiation, perpetuation, or both. The role of target validation, therefore, is to demonstrate the functional role of the potential target in the disease phenotype. The target validation normally require that the; Target is expressed in the disease-relevant cells/tissues, and Target modulation in cell and/or animal models ameliorates the relevant disease phenotype

9. 9 1. mRNA modulation (antisense strategies) Oligonucleotides ribozymes and small interfering RNA (siRNA) 2. Zinc finger printing 3. Transgenic animals Techniques used in target validation

10. 10 Antisense Technologies Antisense technology is a potentially powerful technique which utilizes a strand of nucleic acid (DNA, RNA or a chemical analogue) which are designed to be complimentary to a region of a target mRNA molecule. Binding of the antisense oligonucleotide to the target mRNA results in its inactivation and hence, effectively turning off the gene.

11. 11 Types of antisense strategies Antisense oligonucleotides They usually consist of 15– 20 nucleotides, which are complementary to their target mRNA. They block translation of the mRNA or induce its degradation by RNase H

12. 12 1 st Generation 2 nd Generation 3 rd Generation Here one of the nonbridging oxygen atoms in the phosphodiester bond is replaced by sulphur . The modification improves; • nuclease resistance, • Half life (from 1h to 10hrs), • activate RNase H, 2’-O-methyl and 2’-O-methoxy-ethyl RNA are the most important members of this class. They are less toxic than phosphorothioate DNAs and have a slightly enhanced affinity towards their complementary RNAs DNA and RNA analogues with modified phosphate linkages or riboses as well as nucleotides with a completely different chemical moiety substituting the furanose ring (5 membered ring) have been developed

13. 13 Ribozymes (RNA enzymes) and Small interfering RNA (siRNA) Ribozymes are unique RNA enzymes that can recognize complementary mRNA targets and cleave them at “GUC” sites in a sequence-specific fashion. This makes ribozymes powerful tools for studying the functional consequences of suppressed gene expression. RNAi is a natural process that occurs in many organisms ranging from plants to mammals. In this process, double-stranded RNA or hairpin RNA is cleaved by a Rnase IIItype enzyme called Dicer into small interfering RNA duplex. This then directs sequence-specific, homology-dependent, posttranscriptional gene silencing by binding to its complementary RNA and triggering its elimination through degradation or by inducing translational inhibition.

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15. 15 Zinc Finger Proteins (ZFP) Zinc fingers are small protein domains in which zinc plays a structural role contributing to the stability of the domain. Based on the structural properties in the vicinity of the zinc‐binding site they are grouped into 8 fold groups. C2H2 ‐like finger Gag knuckle Treble clef finger Zinc ribbon Zn2/Cys6‐like finger TAZ2 domain‐like Short zinc‐binding loops Metallothioneins

16. 16 Proteins bind zinc as a cofactor for catalysis or as a structural stabilizer. In zinc fingers, the role of zinc is structural and zinc ions typically do not participate in the function directly. Other parts of a zinc‐binding molecule bear functional importance. • Small protein domains assembled around zinc ions are versatile structural templates that perform various functions. They are involved in; nucleic acid (DNA and RNA) binding, protein–protein interactions, binding small ligands (lipids) and sometimes also possess enzymatic properties Functional properties of ZFP

17. 17 Transgenic technology represents an attractive approach to reduce the attrition rate of compounds entering clinical trials increases the quality of the target and compound combinations making the transition from discovery into development. Transgenic technology influences decision making in target identification, target validation, and can also provide better models for human diseases, Also as model designed to alert researchers early about potential issues with drug metabolism and toxicity. There is a need for in vivo target validation data before large amounts of resource are invested in the potential target. • Considerable efforts are being made to identify rapid, reliable and general tools for in vivo validation, but, so far, only transgenic animals work reliably on a wide range of targets. • A common requirement in the target validation phase is the production of genetically modified animals that either overexpress (gene addition) or no longer possess the target (knockout animals). • These animals provide in vivo functional data on a potential target which are often lacking. Transgenic animals in drug discovery

18. 18 Alzheimer’s disease No animal models existed for the disease before transgenic technology was employed. Today, several transgenic models have been established. These models resemble much of the human pathology and are frequently used in the search for new therapeutic opportunities. Immunisation of Amyloid precursor protein (APP) transgenic mice with the protein Aβ42 before disease became established resulted in disease prevention and, if performed on older animals, inhibited disease progression. This study using transgenic mice suggests that vaccination against Alzheimer’s disease could have potential as a therapeutic approach.

19. 19 Boscha, F., Rosich, L. (2008), The Contributions of Paul Ehrlich to Pharmacology, 171-179. Terstappen, G., Schlupen, C., Raggiaschi, R., & Gaviraghi, G.(2007), Target deconvolution strategies in drug discovery, 891-903. Choe, g., Horvath, S., Cloughesy, T. F., et al. (2003) Analysis of the phosphatidylinositol 3-kinase signaling pathway in gliblastoma patients in vivo. Cancer Res. 63(11), 2742-2746 . REFERENCES :

20. 20 THANK YOU