Combinatorial chemistry and high throughput screening

about lead identification techniques

1. METHODS AND ADVANTAGES IN COMBINATORIAL CHEMISTRY AND HIGH THROUGHPUT SCREENING PRESENTED BY ANJI REDDY Y17MPHPY454 DEPARMENT OF PHARMACOLOGY

2. CONTENTS COMBINATORIAL CHEMISTRY DIFFERENCE BETWEEN TRADITIONAL SYNTHESIS & COMBINATORIAL SYNTHESIS DRUG DISCOVERY TECHNIQUES USED IN COMBINATORIAL CHEMISTRY ADVANTAGES

3. Combinatorial chemistry Combinatorial chemistry comprises chemical synthetic methods that make it possible to prepare a large number (tens to thousands or even millions) of compounds in a single process. These compound libraries can be made as mixtures, sets of individual compounds or chemical structures generated by computer software. Combinatorial chemistry can be used for the synthesis of small molecules and for peptides. Strategies that allow identification of useful components of the libraries are also part of combinatorial chemistry. The methods used in combinatorial chemistry are applied outside chemistry, too

4. Difference Between Traditional Synthesis & Combinatorial Synthesis

5. DRUG DISCOVERY

6. TECHNIQUES USED IN COMBINATORIAL CHEMISTRY solid phase Technique So lid Supp o rt M e th o d P a r all e l S y n thesis Man ua l m e thod A u t om a t ed Mi x ed c omb i n a t or ial S y n thesis Mi x ed & s p li t Comb i n a t or ial S y n thesis Solution phase Technique

7. SOLID PHASE TECHNIQUES Reactants are bound to a polymeric surface and modified whilst still attached . Final product is released at the end of the synthesis Advantages Specific reactants can be bound to specific beads Beads can be mixed and reacted in the same reaction vessel Products formed are distinctive for each bead and physically distinct Excess reagents can be used to drive reactions to completion Excess reagents and by products are easily removed Reaction intermediates are attached to bead and do not need to be isolated and purified

8. Examples of Solid Supports Partially cross-linked polystyrene beads hydrophobic in nature causes problems in peptide synthesis due to peptide folding Sheppard’s polyamide resin - more polar Tentagel resin - similar environment to ether or THF Beads, pins and functionalised glass surfaces

9. Solid phase synthesis: protecting groups A few protecting groups used in solid phase synthesis. For amines. Boc ( t- butoxycarbonyl ) Fmoc (9-fluorenylmetoxy carbonyl) Tmsec (2 [ trimethylsilyl ] ethoxycarbonyl ) For carboxylic acids. Tert Bu ester(t-butyl ester) Fm ester(9-fluronyl methyl ester) Tmse ester(2 [ trimethylsilyl ] ethyl)

10. Parallel Synthesis To use a standard synthetic route to produce a range of analogues, with a different analogue in each reaction vessel, tube or well The identity of each structure is known Useful for producing a range of analogues for SAR or drug optimisation

11. Houghton’s Tea Bag Procedure Each tea bag contains beads and is labelled Separate reactions are carried out on each tea bag Combine tea bags for common reactions or work up procedures A single product is synthesised within each tea bag Different products are formed in different tea bags Economy of effort - e.g. combining tea bags for workups Cheap and possible for any lab Manual procedure and is not suitable for producing large

12. Automated parallel synthesis Automated synthesisers are available with 42, 96 or 144 reaction vessels or wells Use beads or pins for solid phase support Reactions and work ups are carried out automatically Same synthetic route used for each vessel, but different reagents Different product obtained per vessel

13. 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 The identities of the structures in each vessel are not known with certainty Useful for 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 component

14. Mix and Split Method Synthesis of all possible dipeptides using 5 amino acids Standard methods would involve 25 separate syntheses Combinatorial procedure involves five separate syntheses using a mix and split strategy

15. Solution phase Technique It is the modified reaction to accommodate a solid support . Solution phase combinatorial chemistry often lead to a formation of Mixture of product . May helpful for development of Amazing Mixture Problems : difficulty of removing unwanted material purification at each step is necessary other practical problem

16. ADVANTAGES Fast Combinatorial approach can give rise to million of compound in same time as it will take to produce one compound by traditional method of synthesis . Economical A negative result of mixture saves the effort of synthesis, purification & identification of each compound Easy Isolation purification & identification of active molecule from combinatorial library is relatively easy.

17. Drug Discovery Mixed Combinatorial synthesis produces chemical pool. Probability of finding a molecule in a random screening process is proportional to the number of molecules subjected to the screening process Drug Optimization Parallel synthesis produces analogues with slight differences which is required for lead optimization

18. HIGH THROUGHPUT SCREENING

19. CONTENTS INTRODUCTION AND SHORT HISTORY WHAT IS HTS..? INSTRUMENTATION TECHNIQUES AND PROCEDURE IMPORTANCE AND APPLICATIONS OF HTS LIMITATIONS OF HTS

20. INTRODUCTION AND SHORT HISTORY In order to learn about high throughput screening, we should have a general understanding of the drug discovery process. Drug discovery is the process by which new medications or drugs are discovered. It involves the various steps from disease identification, identification and validation of related target(s), development of a lead that can interact with the target and be effective in treating the disease, preclinical and clinical trials and then finally marketing. High throughput Screening was invented by Dr. Gyula Takatsky in 1951; he made the first microtiter plate using Lucite and creating 6 rows of 12 wells in it.

21. WHAT IS HTS..? High throughput screening (HTS) is an experimental process or tool that employs a group of techniques to quickly conduct a very vast number of chemical, pharmacological, genetic, biological tests to identify biomolecular pathways or pharmacological actions. 10,000 – 100,000 compounds can be screened daily. Very vital to drug design and drug discovery process, vital to general scientific and medical research Very valuable to early drug discovery

22. Basically helps to identify a compound that can chemically modify a target This compound is identified as a hit and may be generated to a lead. A Hit is any compound that is confirmed to have binding activity to the target and appears on High throughput screen. It gives the desired effect of the HTS experiment and is confirmed on re-testing. The Lead is the compound with therapeutic or pharmacological activity but suboptimal structure that still requires modification.

23. This process is commonly referred as lead generation or hit to lead (H2L). The lead is further optimized and thus can then go through preclinical and clinical trials and if approved gets marketed. The general procedure of HTS involves testing a solution of different compounds in assay plates called microtiter plates having wells. The ligand or protein or embryo of interest is introduced into these wells containing test solution They are incubated for a short time period. Analysis is done microscopically or by analytical techniques like spectrometry. Compounds showing desired effects are hits.

24. INSTRUMENTATION MICROTITER PLATES (ASSAY PLATES) Plates/containers made of plastic, having spaced wells – up to 384, 1536 or 3456 wells. They would contain solvents (e.g. DMSO + test compounds) They would also contain proteins, cells, etc. to be analysed . Some might be kept empty or contain pure solvents to serve as controls. DETECTORS Diverse spectrometers (fluorescence, mass, NMR, FTIR, etc.), Chromatography (Gas, Liquid, Ion exchange, etc.) and Microscopy (Scanning tunnelling microscopy, atomic force microscopy, confocal microscopy) and Calorimeters.

25. TECHNIQUES AND PROCEDURE TYPES OF HTS: Functional and Non-functional. Functional : Study exactly how the compound interacts with targe Non-functional : To find out if the compound interacts with target or not.

26. PROCEDURE

27. Use of robotics Robotics and automated systems are and impotent component of HTS. They optimize the process and save manpower. Robot arms can be used effectively to transfer microtiter plates to and fro the sampling, incubation and analysing spots.

28. IMPORTANCE AND APPLICATIONS OF HTS Selection of compounds from a vast number synthesized by combinatorial chemistry and other methods. For lead generation for the treatment of a disease. It is an efficient tool in studying biomolecular interactions and pathways. It is highly efficient, fast, accurate and dependable in compound screening Useful in DNA sequencing

29. CONT……………………………………. Useful in toxicology, to study mechanism of action of various drugs and toxins. Study drug-drug interactions and the effects of drugs on metabolizing enzymes. Useful in cytotoxicity assays Useful in genotoxicity assays RECENT ADVANCEMENTS Use of living organisms in HTS to study drug action and identify lead molecules e.g. in Zebra fish and Caenorhabditis elegans . Ultra HTS where above 100,000 compounds are screened at a time; up to 300,000.

30. LIMITATIONS OF HTS High cost Contamination of samples is possible. Analysis of data and selection of relevant data from large moulds of data requires patience,professionalism , dedication and true expertise.

31. BIBLIOGRAPHY An introduction to Medicinal chemistry, rama rao nadendla www.slideshare.net/.../combinatorial-chemistry hts and its applications. www.biotech.nature.com ; NATURE BIOTECHNOLOGY ; Vol 18 ; Supplement 2000.

32. THANK YOU FOR GIVING PRESENTER POSITION TO ME ANJI REDDY