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Discuss the role of combinatorial chemistry with respect to drug design and development.
Drug discovery is a complex process and involves lots of time and money. The main source of biologically active compounds used in the drug discovery has always been natural products, isolated from plants, animals or fermentation sources (1).In past, using the traditional synthetic chemistry the chemists could only make one compound at a time, which then had to undergo isolation and purification through crystallisation, distillation or chromatography which involved a lot of time and energy. To overcome this, new methodologies were developed by researchers in the pharmaceutical industry. This new method reduced the time and costs associated with production of effective and competitive new drugs. Combinatorial chemistry is a technique by which structurally distinct molecules can be synthesised expeditiously and in organised manner immediately applicable for pharmacological assay. Hence large number of chemical analogues can be synthesised using the same reaction conditions and vessel, thus thousands of compounds are produced at a time. This accelerated method of chemical synthesis has a profound impact on all branches of chemistry and especially on drug discovery. This new technology has made the drug discovery process quick and economical to the pharmaceutical companies .
The use of combinatorial chemistry techniques began to spread in early nineties and the since the field had continued to explode (3). Combinatorial chemistry involves the synthesis or biosynthesis of chemical libraries (a family of compounds having a certain base chemical structure) of molecules for the purpose of biological screening, particularly for lead discovery or lead modification. These chemical libraries are made in a systematic and repetitive way by covalent assembly of building blocks (various reactant molecules that build up parts of the overall structure) to give a diverse array of molecules with a common scaffold (parent structure in a family of compounds).The main aim of these combinatorial libraries is to discover a high-affinity, specific molecule that binds to a target protein of interest. These libraries are of two types one is ‘focussed' libraries and the other one is ‘prospecting' libraries. Focussed libraries are also known as targeted, directed, or biased libraries and contain analogs of compound with known biological activity (a ‘lead') and are used to identify alternatives to this structure and to expand structure-activity relationships. In contrast, synthesis of prospecting libraries is a goal directed process; it involves selection of input materials and visions of what can be made with them i.e., structural novelty and diversity being the desired outcome. This design offers a new dimension for creativity and inspiration because the target itself is part of the design (4). Methods of creating combinatorial library were first applied to peptides and oligonucleotides. Since then, the field has been expanded and libraries of small molecules, proteins, synthetic oligomers and oligosaccharides were created (5). The method involved in creating a library is based on the type of library needed. Basically creating a combinatorial library involves three main steps: preparation of a library, screening of the library components and determination of the chemical structures of active compounds.
The most common ways of developing libraries are:
Solid Phase techniques
- Solid support
- The anchor/linker
- Protecting Group Strategy
- Houghton's teabag procedure
- The mix and split method
- High-throughput screening
The main aim in creating these combinatorial libraries is to reduce the number of molecules to be synthesised without decreasing the variety in it, thus enhancing the potential to find a ‘lead' compound rapidly and avoiding molecules which are similar.
Solid Phase Techniques:
This method eliminates the need for the isolation and purification of reaction intermediates. The process of purification is made simple, it is achieved by simply washing the solid support number of times to remove by-products, solvents and residual reagents. This method also enables the use of reagents, catalysts and solvents that are difficult to remove at intermediate stages. Additionally the intermediate reactions may also be driven to completion with excess equivalents of reagents or catalysts.
- Solid Support: The solid support is a polymer resin that has been functionalised along the polymer chain. Wide variety of polymers used some of the examples are polystyrene where the styrene is partially cross-linked with 1% divinylbenzene, Sheppard's polyamide resin and Tentagel an ethyleneglycol/polystyrene resin. Regardless of the polymer resin used the bead needs to be capable of swelling in solvents and still remain stable. Swelling of the beads play an important role in the solid phase synthesis, as the reactions take place in the interior of the beads rather than the surface.
- The Anchor/Linker: It is a molecule that can covalently attach to the polymer chain and form a solid support. The molecular unit contains a reactive functional group with which the starting material in the proposed synthesis can react and hence become attached to the resin. The resulting link must have two properties, first one is stability at the conditions used throughout the synthesis and the second one is ability to release the final residue once the synthesis is finished.
- Protecting Group Strategy: In the solid phase synthesis it is important to protect the functional groups that are not involved hence, the selection of suitable protecting groups is important. The protecting groups should have same properties like the Linker i.e., stability at the reaction conditions throughout the process and ability to release the product at the end of the synthesis. Examples are t-butyloxycarbonyl (tboc) group and 9-fluorenylmethoxycarbonyl (fmoc). The fmoc group is base labile whereas the tboc group is acid labile.
It is a method in which a single reaction product is produced in all the reaction vessels.
- Houghton's Teabag Procedure: This procedure is a manual approach to parallel synthesis and has been used for the parallel synthesis of more than 150 peptides at a time. The resin is sealed in polypropylene meshed containers (teabags) and it is labelled. These teabags are then placed in polyethylene bottles which act as reaction vessels. An example is, to each teabag a different amino acid is added, then the deprotection and washing is undertaken, subsequently another amino acid is added to each reaction vessel and the process is repeated to generate a cleavable peptide sequence. The major drawback in this approach is that it is manual process, and limits the quantity and speed with which new structures can be synthesised.
- The mix and split method: This is the most common method used to generate large libraries (104-106 compounds). In this method a resin support material is divided into a number of equal portions (x) and each portion is made to react individually with a different reagent. Once the reactions are completed, these beads are washed to remove excess reagent and these individual portions are recombined and mixed thoroughly and again divided into portions. This process can be repeated number of times (n). At the end of the process xn numbers of products are produced.
- High-throughput screening (HTS): HTS is the process which allows the researches to carry out several biochemical, genetic or pharmacological tests in short period of time. The main goal of HTS is to accelerate the process of drug discovery by accelerating the screening process. Using this method large number of libraries can be screened in short span of time (6).
Combinatorial chemistry has fundamentally changed the conduct and scope of search for new lead compounds, as it is able to produce new collection of compounds at high speed with molecular diversity which can be subjected to screening. Thus it plays an important role in the discovery of new drugs. Numerous examples of the successful application of both solid and solution phase combinatorial techniques in the generation of active compounds against biologically relevant targets have been documented (7). Combinatorial chemistry plays an important role in discovery of new drugs specially in the areas of infectious diseases caused by bacteria, fungi, parasites and viruses, because these infecting agents easily develop resistance to the available treatments and hence these is always a need for the discovery of new anti-infective drugs. Presently, all widely used antibiotics, including some of the newer agents such as the streptogramins and new generation fluoroquinoles are subjected to bacterial resistance (8). Hence there comes the role of combinatorial chemistry to tackle the resistance problems. Some of the principle ways to challenge these problems are screening of compound libraries, modification of known antibiotics, protection of known classes by resistance mechanism inhibitors and lastly discovery of new agents. And the combinatorial chemistry has enormous impact on all these processes. Several potent anti-viral and anti-fungal drugs have been developed by successful application of solid and solution phase combinatorial chemistry techniques (8). Screening of large mixture based combinatorial peptide libraries led to the successful identification of new anti-microbial compounds. Some of the examples are discovery of trisubstituted furanones, 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazepines, sulfonamido hydroxamic acids, hydrazinyl ureas and 1,5-dialkyl-2,4-dinitrobenzene derivatives as potent antibacterial leads(8). A novel class of antimicrobials that are active against methicillin-resistant Staphylococcus aureus (MRSA), by screening a focussed library of 2-(1-H-indol-3-yl) quinolines was identified. Thus, well designed combinatorial strategies can play an important role in the discovery and rapid optimization of potent new antibiotic leads, and potentiators of anti-microbial action required to overcome bacterial resistance. A group at merck research laboratories has identified selective agonists for the somatostatin receptor using a mixture based small molecule combinatorial library with iterative deconvolution (9). New azoles with extended spectra that included fluconazole-resistant candida strains were discovered with optimized combinatorial libraries (10). An entirely new class of antibiotics, inhibitors of bacterial peptidyl-deformylase has been discovered with the aid of combinatorial chemistry (11).
Combinatorial chemistry and High throughput screening are complementary techniques which have revolutionized the hunt for new drug products and their subsequent testing. Since the end of 1980's the combinatorial techniques have become an industrial standard for the discovery of novel drugs. Some of the advantages of the combinatorial chemistry are, as it is automated equipment it can perform operations 24 hours a day unlike a chemist working in a chemical laboratory. It also performs its task rapidly and has an ability to cope with small amounts of reagents with high precision thus; it works out to be faster and cheaper. It is also environmental friendly as it uses limited reactants. An addition advantage of using this technique is high reproducibility as, the operations are carried out by robotic equipment the chances for experimental errors are reduced. In contrast, there are some drawbacks with these techniques. Though combinatorial chemistry is a potentially speedy process for the discovery of new drugs and other useful compounds, this field is still facing huge criticism for not able to deliver any output to justify the large amounts of investments put in it by the pharmaceutical and chemical industries, academia and the government.
In conclusion, combinatorial chemistry has become an invaluable addition tool in the field of drug discovery. Given that the well-designed combinatorial libraries have been so successful in the pharmaceutical and drug discovery industries, this technology is now being adopted in other field like process development, catalysis and material sciences. In view of the fact that the development of pharmaceutical drugs takes long time unfortunately, there are no examples yet of marketed drugs discovered by combinatorial methods. If it is applied sensibly it can deliver number of drug-like compounds in a timely fashion. However there is an urgent need for new drug discoveries in some of the therapeutical areas like oncology and infective diseases. As it is evident that many drugs used today have their origin in natural products these techniques of combinatorial chemistry should be further applied to natural products and generate newer libraries. Though this field had a dramatic influence on the drug discovery process, it is still at its infacy and still has to go a long way before its full potential can be realised.