_Lead_identification_and_optimization_.pptx

M pharmacy pharmacology

1. Lead identification and optimization Submitted by: Ayodhya Paradhe Submitted to : Dr. Mandade Sir M pharm 1 st year (Pharmacology) (Pharmacology Department) Sudhakar Rao Naik Institute of Pharmacy Pusad

2. Contents Introduction Lead identification methods Lead optimization

3. Introduction Lead compound: Once a target and testing system has been chosen, the next is to find a lead compound that shows the desired pharmaceutical activity. Lead compound is a new chemical entity that could potentially be developed into a new drug by optimizing its beneficial effects to treat diseases and minimize side effects. These are typically used as starting points in the drug design to give new drug entities. These drug design strategies can be used to improve the compounds pharmacodynamic and pharmacokinetics properties. The resulting compounds from drug design go through a series of preclinical studies and become clinical candidates if the compounds don’t exhibit adverse effects or toxic effects during in vivo and vitro studies. After going through marketing obstacles and clinical trials, compounds that pass are released in the market as new drug entities and these are generally monitored for safety after their released on the market.

4. Ways to find a lead compound: 1. nature: bacteria, molds , plant extract Eg. natural products- ephedrine, propranolol plant: morphine, cocaine, digitalis etc. 2. De novo: scientist can also create molecules from scratch computer modelling 3. High throughput screening: test thousands of compounds against the target to identify any that might be promising 4. biotechnology: scientist can genetically engineer living systems to produce disease-fighting biological molecules

5. Properties of lead: 1.Pharmacodynamic properties: efficacy, potency, selectivity 2. Physicochemical properties: water solubility, chemical stability 3. Chemical optimization potential: ease of chemical synthesis and derivatization 4. patentability lead identification: Identification of small molecule modulators of protein function and the process of transforming into high-content lead series are key activities in modern drug discovery. Lead compound is first foothold on drug discovery ladder It takes more efforts to make lead compound into drug candidate.

6. Methods of lead identification: 1. Random screening 2. Non-random screening 3. Combinatorial chemistry 4. High throughput screening(HTS) 5. In silico lead discovery techniques 1.Random screening: This methods was followed before 1970. All compounds including synthetic chemicals, natural products of plants marine and microbial origin form a given series are tested. Insite of budgetary and manpower overuse, this method may be use to discover drugs or leads that have unexpected activities. Antibiotics like, streptomycin and tetracyclines were found out by this method.

7. 2. Non-random screening method: After 1970 this method was used. it is a modified form of random screening that was developed because of budgetary and manpower restrictions. In this method only such compounds having similar structural skeletons with that of lead are tested. 3. combinatorial chemistry: combinatorial chemistry is a technique by which large numbers of different but structurally similar molecules are produced rapidly and submitted for pharmacological assay. Primary objective of this method is reduce the cost and to synthesize large number of molecules. This technique uses the same reaction conditions with same reaction vessels to produce a large range of analogues. Combinatorial chemistry encompasses many strategies and processes for the rapid synthesis of large, organized collection of compounds called libraries. The collection is tested for the biological activities.

8. Finally the active compound is identified and made in quantity a a single compound. Thus this method approaches has two phases: 1.Making a library 2. Finding the active compound . Screening mixtures for biological activity has been compared to finding a needle in haystack. Principle of combinatorial chemistry: The basic principle of this is to prepare a large range of different compounds at same time instead of synthesizing compounds in a conventional one in a timely manner and identify the most promising compound for further development by high throughout screening. The characteristics of combinatorial synthesis is that different compounds are generated simultaneously under identical reaction conditions, in a systematic manner so that ideally the products of all possible combinations af a given set of starting materials ( building blocks) will be obtained at once .

9. The co llection of these finally synthesized compounds is referred as a combinatorial library. The library is then screened for useful properties and the active compounds are identified. combinatorial chemistry within design: therapeutic target combinatorial chemistry lead discovery can impact here Lead optimization development candidate drug

10. Types of combinatorial synthesis 1. Solid phase techniques 2. Parallel synthesis a) Houghton’s Tea Bag procedure b) Automated parallel synthesis c) Automated parallel synthesis of all 27 tripeptides from amino acids 3. Mixed combinatorial synthesis 4. Solution phase synthesis 1. Solid phase technique: In this the compound library has been synthesized on a solid phase such as resin bead. Starting compound attached to an insoluble resin bead, reagents are added to the solution in excess and the resulting product can be isolated by simple filtration, which traps the beads while the excess reagents are washed away. Steps involved: 1. Attachment of starting material to an inert solid/ resin bead 2. Addition of excess reagents in solution 3. Separation of product from resin bead by simple filtration 4. Cleavage and isolation of attach product from the resin bead

11. Requirements: a) A crossed linked, insoluble, polymeric material that is inert to condition of synthesis b) Linker or anchor; a bond linking the substrate to linker. The bond must be stable to the reaction condition used in synthesis. Linking the substrate to solid phase that permits the cleavage of some or entire product from the solid support during synthesis for analysis of the extent of reaction and ultimately to give the final product of interest. Eg. Merrified resin, hydroxymethyl resin, amino methyl resin, link amide resin c) A chemical protection strategy (i.e. protecting group) to allow selective orthogonal and de-protection of reactive groups in the monomers. Eg. Benzyl carbonyl (Z) group, t- butoxy carbonyl group for protection of amino group, benzyl ester and t- butyl ester for protection of carboxyl group. Examples of solid support: Partially crossed linked polystyrene beads: polystyrene cross linked with divinylbenzene, Sheppard’s polyamide resin: more polar Tentagle resin Beads, pins, functionalized glass surfaces

12. Advantages: Specific reactants bound to specific beads Beads can be mixed and reacted in the same reaction vessels Excess reagents and products are easily removed Reaction intermediates are attached to bead and do not need to be isolated and purified Polymeric support can be regenerated and reused after cleaving the product Improved reaction yield Disadvantages: Optimal reaction conditions for solid phase synthesis can be difficult to determine and developing and these are more time consuming There is limited range of chemistry available for attachment to the resin in solid phasei.e . some molecules don’t attached to well and bead. Difficult to monitored the progress of reaction when the substrate and product ae attached to the solid phase. Assessment of the purity of the resin attached intermediates is also difficult. Removal of product from bead can be damaging to product if not careful.ss

13. 2. Parallel synthesis: To use a standard route to produce a range of analogues with a different analogues in each reaction vessel, tube or well. The identity of each structure is known. Useful for producing a range of analogues for SAR drug optimization. 3. Mixed combinatorial synthesis: to use a standard synthetic route to produce a large variety of different analogues where each reaction vessel or tube contains a mixture of products. Useful to finding a lead compound. Capable of synthesizing large numbers of compounds quickly. Each mixture is tested for activity as the mixture. Inactive mixtures are stored in combinatorial libraries. active mixtures are studied further to identify active compounds.

14. 4. Solution phase synthesis: In this synthesis involves conducting chemical reactions simultaneously in well-ordered sets of reaction vessels in solution. Most ordinary synthetic chemistry takes place in the solution phase. The use of this technique has been explored as an alternative to solid-phase chemistry approaches for the preparation of arrays of compounds in drug discovery. In this use a soluble polymer support for the product PEG is a common vehicle which is used in solution phase synthesis it can be liquid or solid at room temperature and shows varying degrees of solubility in aqueous and organic solvents. Another support polymer Teflon is used. When this is used as a soluble support for synthesis the resulting product can be easily separated from any organic solvents.

15. 4. High throughput screening(HTS): History: High throughput screening invented by Dr. Gyula Takatsky in 1952: he made the first microtiter plate using Lucite and created 6 rows and 12 wells in it,. It useful in the process that lead to lead identification. HTS as an experimental process or tools that employ the group of techniques to quickly conduct a very vast number of chemical, pharmacological, genetic, biological test to identify biomolecular pathways or pharmacological actions. It is drug discovery process widely used in the pharmaceutical industry. 10,000- 100,000 compounds screened daily. Very valuable to early drug discovery. Basically helps to identify compounds that can chemically modify a target. The goal is to identify ‘hit’ or ‘lead’. It is useful for discovering ligands for receptors, enzymes, ion channels or other pharmacological targets.

16. Types of HTS: 1. functional: study exactly how the compound interacts with target 2. non-functional: to find out if the compound interacts with the target or not. Instrumentation: 1. Microtiter plates( assay plates): Plates/containers made of plastic, having spaced wells- up to 384,1536 or 3456 wells. They would contain solvents. ( DMSO+ test compound) also contain proteins, cells etc. to be analysed Some might be kept empty or contain pure solvents to serve as control. 2. detectors: spectroscopy- fluorescence spectroscopy, NMR, FTIR, absorption and luminescence, light scattering, mass spectroscopy b) chromatography- GC, TLC, HPLC, ion exchange chromatography, reverse phase chromatography, affinity chromatography

17. c) Calorimetry- isothermal titration calorimetry(ITC), differential scanning calorimetry(DSC) d) microscopy- scanning Tunneling microscopy, atomic force microscopy, confocal microscopy Uses of HTS: To screen microarrays: DNA chips RNA chips Protein chips To screen for all kinds of novel biologically active compounds: Combinatorial libraries Biological libraries Natural products

18. Procedure:

19. steps involved IN HTS: Add DMSO or any other solvents in microtiter plate well and label them add test solution in some, leave others to serve as control The test should be labeled incubated for period of time with fluorophores or dyes e.g. Almar blue observe microscopically Detect change in fluorescence And view on scree

20. Importance and application of HTS Selection of compounds from a vast number synthesized by combinatorial chemistry and other methods For lead generation for the treatment of disease Highly efficient, fast, accurate and dependable in compound screening Useful in toxicology, to study the mechanism of action of various drugs and toxins Study drug-drug interaction and effects of the drug on metabolic enzymes Useful in cytotoxicity, genotoxicity assay

21. 5. In Silico Lead Discovery Techniques In silico is an expression used to mean performed on computer or via simulation In silico drug designing is defined as the identification of the drug target molecules by employing bioinformatics tools Types: Ligand-based drug discovery structure-based drug designing 1. Ligand-based drug discovery: Ligand-based drug design relies on knowledge of other molecules that bind to the biological target of interest Used to derive a pharmacophore pharmacophore

22. 2. Structure-based drug design: Structure-based drug design relies on knowledge of three dimensional structure of the biological target obtained through methods such as : X-ray crystallography NMR spectroscopy Homology modelling Using the structure of the biological target, drugs that are predicted to bind with to the target may be designed using: Interactive graphics The intuition of a medicinal chemist Automated computational procedures

23. LEAD OPTIMIZATION Lead optimization is the process that begins with a compound that shows a potential biological action and confirms with the identification of the best compound Molecules are chemically modified and characterized to make a compound with desired properties to become a drug Leads are optimized for efficacy and potency in vitro and in vivo, physicochemical properties, pharmacokinetic properties, and toxicological aspects This stage is a major vault in drug discovery it contributes to turning a biologically active chemical into an effective and safe drug in the drug discovery process.

24. Structural variation of the lead to obtain a drug with desired profile. Objectives of molecular manipulation- To developed substitutes for existing bioactive compounds eg. Hormomes , vitamins, neurotransmitters. To change the spectrum of activity of lead- To obtain antagonists from agonist. To separate activities- androgenic steroids anabolic steroids sulphonamides antidiabetics, diuretics To combine the action of different drugs- action of oxytocin + vasopressin oxypressin To eliminate side effect - glucocorticoids: cortisone prednisone dexamethasone mineralocorticoid activity 0.8 0.6 0s

25. 3. To modulate the pharmacokinetics of lead- a) To decrease sensitivity to degrading enzymes- acetylcholine methacholine To modify drug distribution- methylatropine – quaternary compound, no CNS activity sulphonamides for treatment of intestinal infections- phthalsulphacetamide To modify time- conc. Relationship- iv therapy – hydrocortisone sod. succinate

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