DR. RITESH P. BHOLE Associate Professor Dr. D. Y. Patil Institute of Pharmaceutical

1. DRUG DESIGN AND DISCOVERY A NEED OF HOUR DR. RITESH P. BHOLE Associate Professor Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research Pimpri, Pune - 411 018

2. OR SCENARIO - 1 SCENARIO - 2

3. What happen in past ? • Ancient times - medicines were herbs and poisons • Serious efforts were made to isolate and purify the active principles - after the mid - nineteenth century. • A large variety of biologically active compounds were obtained and structures determined (e.g. morphine, cocaine, quinine etc.) • Natural products became the lead compounds • No real design or reason.

4. WHAT DRUG DESIGN AND DISCOVERY IS AND WHY IT'S IMPORTANT The process of the design/discovery of drugs typically involves understanding the character of targets (e.g. enzyme, cell, tissues, etc) related to the disease e.g. COX enzyme has to target if we want to treat Inflammation. setting-up the concept of drug design, providing lead compounds (via traditional medicines, natural products, biological macromolecules, compound libraries, computational chemistry, etc.) lead optimization by means of analyzing structure-activity-relationships. A deep understanding of this process in addition to the mode of action at a molecular level

5. DRUG DISCOVERY Drug Discovery is the process through which potential new medicines are identified. 3

6. PROCESS OF DRUG DESIGN AND DRUG DISCOVERY

7. PURPOSE OF DRUG DISCOVERY • Drug discovery initiates because there is a disease or clinical condition without suitable medical products available. • Rare diseases or orphan diseases.

8. TARGET IDENTIFICATION • TARGET • It is a cellular or molecular structures involved in pathology which are responsible for disease. They may be - • Receptors • Enzyme • Nucleic acid • Hormone • Ion Channel • TARGET IDENTIFICATION • It is the process of identifying the direct molecular target i.e protein, nucleic acid. • It is aimed at finding the efficacy target of a drug. • It is the first step in drug discovery 5

9. TOOLS FOR TARGET IDENTIFICATION Genomics Proteomics Tools for Target Identification Bioinformatics

10. TARGET VALIDATION • Target validation is the process by which the predicted molecular target - for example protein or nucleic acid - of a small molecule is verified. • Target validation can include knockdown or over expression of the presumed target. Tools for Target Validation Antisense technologies Antisense Oligonucleotides siRNA 15

11. LEAD IDENTIFICATION • Screening of natural products • The plant kingdom, The microbial world, The marine world , Animal sources, Venoms and toxins • Medical folklore • Screening synthetic compound libraries • Existing drugs ‘me too’ drugs eg:- captopril enhancing a side effect, eg:- sulfonamides & sulfonylureas • Starting from the Natural Legend or Modulator • Natural legends for receptors - Adrenalin, nor adrenalin • Natural substrates for enzymes – enkephalins • Enzyme products as lead compounds - Product of an enzyme catalysed reaction • L-benzyl succinic acid (Carboxypeptidasecatalysed hydrolysis) • Natural modulators as lead compounds

12. LEAD IDENTIFICATION • Combinatorial synthesis • It is an automated solid phase procedure aimed at producing as many different structures as possible in as short a time as possible. • Computer aided design • Serendipity and the prepared mind Eg: Cisplatin, Ampicillin • Computerised searching for structural database Database mining

13. LEAD OPTIMIZATION • Identify structure activity relationships (SARs) • Identify the Pharmacophore • Drug optimization : strategies in drug design

14. STRUCTURE ACTIVITY RELATIONSHIP • The aim here is to discover which parts of the molecule are important to biological activity and which are not. • It is important to identify the binding roles of different groups.

15. IDENTIFICATION OF PHARMACOPHORE The pharmacophore summarizes the important binding groups which are required for activity and their relative positions in space with respect to each other

16. DRUG OPTIMIZATION Strategies in drug design • VARIATION IN SUBSTITUENTS • Alkyl substituents • Adrenaline, methyl group substituted with isopropyl gives isoprenaline • Aromatic substitutions • Benzopyran substituted with sulfonamides, increases antiarrythmic activity • EXTENSION OF THE STRUCTURE • Chain extension / contraction Eg. Pencillin

17. LEAD OPTIMIZATION FOR REDUCING THE TOXICITY • PRODRUGS • Prodrugs to improve membrane permeability - enalapril enalaprilat • Prodrugs to prolong drug activity Azathioprine6 - mercaptopurine • Prodrugs masking drug toxicity and side effects Aspirin salicylic acid • Prodrugs to lower water solubility Palmitate ester of Chloramphenicol

18. WHY PRELICNICAL TESTING • Whether a drug will move on to studies in humans • Designing phase I clinical trials • Help to identify criteria for evaluating safety in humans • Detect overt toxicity • Identify, describe and characterize hazards reversible? • Clinically monitor able? • Establish dose-response estimation of pharmacology and toxic effects • Assess drug distribution to organ systems • Identify metabolic, kinetic and elimination pathways • Assess carcinogenicity, reproductive toxicity and teratogenic potential

19. PRELICNICAL TESTING - MAJOR CONSIDERATION • Medical uses/needs forsubstance • Commercialpotential • Feasibility for mass production (manufacturingcosts)

20. PHASE I : Designed to verify safety & tolerability of the candidate drug in humans Typically takes 6-9 months PHASE II : To determine effectiveness and further safety of the candidate drug in humans Generally takes 6 months to 3 yrs PHASE III : Expanded testing Takes 1-4 yrs TESTING ON HUMAN VOLUNTEERS

21. New Approaches used in Drug Design and Discovery

22. transgenic animals

23. INTRODUCTION A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Transgenesis is the process by which mixing up of genes takes place. Foreign genes are inserted into the germ line of the animal, so it can be transmitted to the progeny. Transgenic technology has led to the development of fishes, live stock and other animals with altered genetic profiles which are useful to mankind.

24. INTRODUCTION First transgenic animal was a ‘Supermouse’ created by Ralph Brinster (U Pennsylvania) and Richard Palmiter (University of Washington) in 1982. It was created by inserting a human growth hormone gene in mouse genome. The offspring was much larger than the parents. Mouse - common transgenicexpt. Other animals include pig, goat, cow, sheep, fishetc.

25. PRODCUTION OF TRANSGENIC ANIMAL - METHODOLOGY • STEP 1 : • Construction of atransgene, which is made-up of 3 parts, • Pomoter • Gene to beexpressed • Terminationsequence • STEP 2 : • Introduction of foreign gene into theanimal • Pronuclear microinjectionmethod • Embryonic stem cellmethod.

26. STEP 3 : • Screening of transgenic positives • Transgenic progenies are screened by PCR to examine the site of incorporation of the gene • Some transgenes may not be expressed if integrated into a transcriptionally inactive site. • STEP 4 : • Further animal breeding is done to obtain maximal expression • Heterozygous offsprings are mated to form homozygous strains.

27. PROBLEMS • Multiple insertion - too much proteins • Insertion into an essential gene - lethality • Insertion into a gene leading to gene silencing • Insertion into a different area can affect the gene regulation

28. WORKING EXAMPLE TARGET IDENTIFICATION Suppose we identify disease i.e. Alzeimer - Translated this disease model into animals - Animal model of AD ( Streptozocin used to induce - beta amyloid protein in hippocampus… Target identification by proteomics and genomics - Over expression of APP protein is responsible for disease -

29. WORKING EXAMPLE • TARGET VALIDATION • Validate that really APP is responsible for AD. • Deliver mutant APP gene into animal • Progenies having mutant APP gene overexpressed • Target validated

30. Some examples of transgenic animals

31. FISH • SUPER FISH • Increased growth and size • Growth hormone gene inserted into fertilized egg. • Transgenic salmon grows about 10 - 11 times faster than normal fish. • GLO FISH • GM freshwater zebra fish (Daniorerio) • Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish.

32. MOUSE • ALZHEIMER’SMOUSE • In the brain of Alzheimer’s patients, dead nerve cells are entangled in a protein called amyloid. • Mouse made by introducing amyloid precursor gene into fertilized egg of mice. • ONCOMOUSE • Mouse model to study cancer • Made by inserting activated oncogenes. • SMART MOUSE • Biological model engineered to overexpress NR2B receptor in the • synaptic pathway. • This makes the mice learn faster like juveniles throughout their lives.

33. RABBIT • Alba, the EGFP (Enhanced Green Florescent protein) bunny • Created in 2000 as a transgenicartwork. MONKEY • ANDi was the first transgenic monkey, born in 2000. • “ANDi” stands for “inserted DNA” spelled backwards. • An engineered virus was used to insert the harmless gene for green fluorescence protein (GFP) into ANDi’s rhesus genome. • ANDi proves that transgenic primates can be created, and can express a foreign gene delivered into their genome.

34. INPORTANCE OF TRANSGENESIC ANIMALS • MEDICAL IMPORTANCE • Disease model • Bioreactors for pharmaceuticals • Xenotransplantation (the process of grafting or transplanting organs or tissues between members of different species.) • AGRICULTURAL IMPORTANCE • Disease resistant animals • For improving quality and quantity of milk, meat, eggs and wool production • INDUSTRIAL IMPORTANCE • Toxicity sensitive transgenic animals to test chemicals. • Spider silk in milk of goat

35. CONCLUSION • The discovery and development of new medicines is a long, complicated process. • Each success is built on many, many prior failures. • Advances in understanding human biology and disease are opening up exciting new possibilities for breakthrough medicines.

36. SOME IMPROTANT LINKS SOME SOFTWARES https://www.click2drug.org/ www.pdb.org https://www.biopharmatrend.com http://www.bindingmoad.org/ http://zinc15.docking.org/ ACD Chemsketch Autodock Swissdock E Dragon (QSAR)

37. THANK YOU

38. ANY QUESTION