INTRODUCTION:

This is the process whereby “Hits” from screening are transformed into “lead” that can be used for “Lead Optimisation”. During the early stages of drug discovery for a certain disease, the study of molecular mechanism is essential. These studies include identifying the cellular and genetic factors involved in the disease.⁴ In order to ensure that the biological target is involved in the disease, in Vitro (Isolated cell), and in Vivo (Animal model), these all process is also known as target validation, the combination of all in vivo, in vitro, and in silico (computer based) studies are also called as lead Identification.⁵

“When small molecules are hit on the high throughput screen (HTC) are evaluated and undergo limited optimisation to identify lead compound”.²

Process-

Some important steps are used for the identification of target.

Fig: 1.1

Combinational Chemistry:

The Combinational chemistry or combichem is a chemical synthetic method that make a possible to formed a many (tens to thousands or millions) compounds in a single process. In this process, a mixture is prepared of multi components/sets of individual compound and structure is generated by computer software. This branch of chemistry is used for the synthesis of small molecule and peptides.¹

Principle:

The “Combinational Chemistry is based on the large no. of structurally distinct molecule may be synthesised at a time in a reaction and submitted for Pharmacological assay”. In this technique the less time and cost are consume for the producing effective, marketable, and competitive new drugs.⁸ See figure 1.2

Figure: 1.2 Combinational Chemistry

A growing trend toward the synthesis of complex natural-product-like libraries, including the carbohydrate-based libraries, An increased focus on “phase trafficking” techniques are used for integrating synthesis with purification, The main goal of combinatorial chemistry able to synthesis, purify, chemically analyze, and biologically test all the structures in the library, using as few synthetic experiments as possible, it was firstly applied in the synthesis of peptides.⁹

Types:

According to their specification and up gradation, there are of two mainly types:

· Solid Phase Combinatorial Chemistry

· Solution Phase Combinatorial Chemistry

1. Solid Phase Combinatorial Chemistry:

In this type of combinatorial chemistry, the reagents or products are attached to solid supports such as polystyrene bead. These beads are easily available and very easily purify the product by filtration.¹⁰

Solid phase chemistry has better than the solution-phase. Like as, in solid-phase synthesis, large excesses of reagents can be used to drive reactions to completion; these excess reagents can then be removed at the end of the reactions by filtration and washing, and it too easy separation of reagents and products, solid phase chemistry can be automated more easily than solution chemistry.¹¹

Characteristics of solid used:

The using polymer beads are ranging from 10 to 750µm in diameter and these solids should have some properties as Chemically inert, Physically stable, Swelling ability, functional group attached on suitable site with appropriate covalent attachment, etc.

The compounds to be synthesized are not attached directly to the polymer molecules.²⁴ They are usually attached by using a linker moiety that enables attachment in a way that can be easily reversed without destroying the molecule that is being synthesized and allow some room for rotational freedom of the molecules attach to the polymer.¹⁷ Some solids are used in SPCC-

· Polystyrene resins (it is suitable for non-polar solvents)

· Tenta Gel resins (it suitable for polar solvents)

· Polyacrylamide resins (Better for polar solvent)

· Glass and ceramic beads (Resist high temperature)

2. Solution Phase Combinatorial Chemistry:

Mostly synthetic chemistry is takes place in solution phase. The use of solution phase techniques has been explored as an alternative to solid-phase chemistry because in this preparation the constraints of solid phase and it easily purified. In solution phase synthesis we use soluble polymer as 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 show varying degrees of solubility in aqueous and organic solvent.²³ By converting one OH group of PEG to methyl ether (MeO-PEG-OH) it is possible to attached a carboxylic acid to the free OH and use in solution phase combinatorial synthesis. When the liquid Teflon is used as a soluble support for synthesis the resulting product can be easily separated from any organic solvent.¹⁹

Synthesis of Polymer by Solution Phase Combinatorial Chemistry:

Reaction of the reagent with a carboxylic acid in the presence of an activating agent afforded the polymer bound activated ester which was reacted with amines to liberate the amide in solution and formed 1-hydroxybenzotriazole. Supported electrophilic, nucleophilic or ionic reagents used to remove impurities from solution have been termed scavenger reagents; polymer supported quenching reagents (PSQ) or complementary molecular reactivity/molecular recognition polymer (CMR/R polymer).²² Use of such reagents provides a versatile counterpart to the approach. Booth and Hodges utilised a high loading amine resin derived from chloromethyl polystyrene and tris (2 aminoethyl) amine in the preparation of ureas, thioureas, sulphonamide.³

Synthesis of Thiohydantoins:

Sim and Ganesan developed a one-pot three component synthesis of thiohydantoins using the reductive amination of amino esters with aromatic aldehydes and sodium tri acetoxyborohydride followed by the reaction with an isocyanate in the presence of triethylamine.⁶ The thiohydantions were isolated by an aqueous work-up protocol which incorporated the addition of glycine to convert unreacted reagents into water soluble materials. The methodology was used in the preparation of an array of 600 discrete compounds.^7,8

Uses:

Some important use of combinational chemistry are-

· Used to synthesise a large no. of different compounds and screening for biological activity.

· Result in a “combinational library”.

· Combinational chemistry involves the rapid synthesis of a large no. of different but structurally related molecules.²¹

Functions:

Some important function of both Combinational and Conventional are:

Conventional Combinational

One molecule at a time Many molecule at a time

Make→Purity→Test Make→Test→Purity

Hundreds of Molecules Thousands of molecules

A month A Half Month

Slower lead Generation Faster lad Generation

High risk of failure Low risk of Failure

↓ ↓

Synergy

↓

Lead Identification

High Throughput Screening (HTS):

High-Throughput Screening (HTS) is an approach to drug discovery that has gained widespread popularity over the last two decades and has become a standard method for drug discovery in the pharmaceutical industry. It is also used to characterize metabolic, pharmacokinetic and toxicological data about new drugs. It involves the fully automated robotic systems, enables testing of large numbers of compounds daily for different activities in different areas of biology and chemistry. HTS plays an essential role in the drug discovery process.¹²

Through the HTS more than 100,000 samples per day are evaluated because is better than other screening due to its simplicity, rapidness, low cost, and high efficiency. Identification of good hits using HTS can minimize the time span of drug discovery.¹¹

TYPES OF HIGH THROUGHPUT ASSAYS:

HTS are mainly categorised into two mainly types-

· Biochemical Assay

· Heterogeneous assays

· Homogeneous assays

Cell Based Assay:

Biochemical Assay:

Biochemical assays are receptor, protein or enzyme-based assays uses the particular target in a purified form. Biochemical assays are most frequently carried out using scintillation proximity assay (SPA), radiometric, colorimetric fluorescence detection techniques. The biological responses measured in HTS assay span isolated biochemical system containing purified receptors or enzyme to signal transduction pathway and complex network functioning in cellular environment. Biochemical assays are further divided into two parts-^{14, 15}

A. Homogeneous assay:

In homogeneous assay measurement are based on the distinct physical/chemical properties of analyte, or interaction between analyte and surrounding environment. It is a single step process; reagent may be added at single stage or in multiple steps. It only involves usual steps like fluid addition, incubation and reading.^[30] It can be coupled with different detection technique fluorescence, radiometric etc for HTS. The main advantage of homogeneous assay is its simplicity because the minimum step in an assay.¹³

B. Heterogeneous assays:

The Heterogeneous assays involves additional steps like filtration, centrifugation etc. that separates component to be measured from the rest of component which may interfere in assay.²⁹ Due to higher steps it becomes complicated. Heterogeneous assay are performed mainly when homogeneous assay fails or high signal to background ratio is required.²⁰

Applications:

i) Determination of molecular interaction.

ii) Study conformation changes.

iii) Concentration and aggregation measurements, diffusion analysis.

iv) Can be used in binding assays and enzymatic assays.

v) Structural and molecular dynamics of fluorescent proteins in vivo and in vitro.

vi) This has been used to quantify biochemical properties such as protein denaturation, and attachment of proteins to nucleic acid etc.

vii)Used to study receptor/ligand studies, Tyrosine Kinase Assays etc.²⁵

Figure: 1.6 Cell Based Assay

Cell Based Assay:

The cell-based assay is defined as any assay that take place within living cells. Cell-based assays for HTS can be classified under following classes-

1. Second messenger assays:

It monitors signal transduction from activated cell-surface receptors. Second messenger assays typically measure fast, transient fluorescent signals that occur in matter of seconds or milliseconds. Many fluorescent molecules are known to respond to changes in intracellular Calcium ion concentration, membrane potential and various other parameters, hence they are used in development of second messenger assays for receptor stimulation and ion-channel activation.^16-27

2. Reporter gene assays:

It monitors cellular responses at transcription/translation level. It indicates the presence or absence of a gene product that in turn reflects changes in a signal transduction pathway.²⁸ Plasmids are typical reporter genes employed. It is used in vitro study for generation and screening of combinatorial protein library in array format. This studied employed virtues of polymerase chain reaction (PCR) and in vitro coupled reporter gene assay.¹⁸

3. Cell proliferation assays:

It monitors the overall growth/no growth responses of the cell to external stimuli. These are quick and easy to be employed for automation.

CONCLUSION:

During the early stages of drug discovery for a certain disease, the study of molecular mechanism of every drug is essential for generation of a new moiety. These studies include identifying the cellular and genetic factors involved in the disease. In order to ensure that the biological target is involved in the disease, in Vitro and in Vivo, these all process is also known as target validation, the combination of all in vivo, in vitro, and in silico (computer based) studies are also called as lead Identification. For best result and generation of new update moiety after use these techniques.

DECLARATION OF INTEREST:

The authors report no conflict of interest. The authors, alone are responsible for the content and writing of the paper.

REFERENCES:

1. Gordon, E. M., Barrett, R. W., Dower, W. J., Fodor, S. P. A., Gallop, M. A. Applications of Combinatorial Technologies to Drug Discovery: 2. Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions. J. Med. Chem. 1994, 37, 1385–1401.

2. Schafffrath, M., von Roedern, E., Hamley, P., Stilz, H. U. High-Throughput Purification of Single Compounds and Libraries. J. Comb. Chem. 2005, 7, 546–553.

3. Ohlmeyer, M. H., Swanson, R. N., Dillard, L. W., Reader, J. C., Asouline, G., Kobayashi, R. Complex Synthetic Chemical Libraries Indexed with Molecular Tags. Proc. Natl. Acad. Sci. 1993, 90, 10922–10926.

4. Erlandson, D. A., McDowell, R. S., O’Brien, T. Fragment Based Drug Discovery. J. Med. Chem. 2004, 47, 3463–3482.

5. Carr, R. A. E., Congreve, M., Murray, C. W., Rees, D. C. Fragment-Based Lead Discovery: Leads by Design. Drug Disc. Today. 2005, 10, 987–992.

6. Krishnamurthy, V. M., Semetey, V., Bracher, P. J., Shen, N., Whitesides, G. M. Dependence of Effective Molarity on Linker Length for an Intramolecular Protein-Ligand System. J. Am. Chem. Soc. 2007, 129, 1312–1320.

7. Shuker, S. B., Hajduk, P. J., Meadows, R. P., Fesik, S. W. Discovering High-Affinity Ligands for Proteins: SAR by NMR. Science. 1996, 274, 1531–1534.

8. Hajduk, P. J., Sheppard, G., Nettesheim, D. G., Olejniczak, E. T., Shuker, S. B., Meadows, R. P, Discovery of Potent Nonpeptide Inhibitors of Stromelysin Using SAR by NMR. J. Am. Chem. Soc. 1997, 119, 5818–5827.

9. Congreve, M. S., Davis, D. J., Devine, L., Granata, C., O’Reilly, M., Wyatt, P. G., Jhoti, H. Detection of Ligands from a Dynamic Combinatorial Library by X-ray Crystallography. Angew. Chem. Int. Ed. 2003, 42, 4479–4482.

10. Lyne, P. D. Structure-Based Virtual Screening: An Overview. Drug Disc. Today. 2002, 7, 1047–1055.

11. Irwin, J. J., Shoichet, B. K. ZINC: A Free Database of Commercially Available Compounds for Virtual Screening. J. Chem. Inf. Model. 2005, 45, 177–182.

12. Forino, M., Jung, D., Easton, J. B., Houghton, P. J., Pellecchia, M. Virtual Docking Approaches to Protein Kinase B Inhibition. J. Med. Chem. 2005, 48, 2278–2281.

13. Tervo, A. J., Kyrylenko, S., Niskanen, P., Salminen, A., Leppänen, J., Nyrönen, T. H., An in Silico Approach to Discovering Novel Inhibitors of Human Sirtuin Type 2. J. Med. Chem. 2004, 47, 6292–6298.

14. Kellenberger, E., Springael, J.-Y., Permentier, M., HachetHaas, M., Galzi, J.-L., Rognan, D. Identification of Nonpeptide CCR5 Receptor Agonists by Structure-Based Virtual Screening. J. Med. Chem. 2007, 50, 1294–1303.

15. Cole, C. J., Pfund, W. P. Permeability Assays. Mod. Drug Disc. 2000, 3, 73–76.

16. Li, A. P. Preclinical in Vitro Screening Assays for DrugLike Properties. Drug Disc. Today. 2005, 2, 179–185.

17. Wunberg, T., Hendrix, M., Hillisch, A., Lobell, M., Meier, H., Schmeck, C. Improving the Hit-to-Lead Process: Data-Driven Assessment of Drug-Like and Lead-Like Screening Hits. Drug Disc. Today. 2006, 11, 175–180.

18. Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Del. Rev. 1997, 23, 3–25.

19. Teague, S. J., Davis, A. M., Leeson, P. D., Oprea, T. The Design of Leadlike Combinatorial Libraries. Angew. Chem. Int. Ed. 1999, 38, 3743–3748.

20. Veber, D. F., Johnson, S. R., Cheng, H.-Y., Smith, B. R., Ward, K. W., and Kopple, K. D. Molecular Properties That Influence the Oral Bioavailability of Drug Candidates. J. Med. Chem. 2002, 45, 2615–2623.

21. Edwards, M. P., Price, D. A. Role of Physiochemical Properties and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks. Annu. Rep. Med. Chem. 2010, 45, 381–391.

22. Nassar, A.-E. F., Kamel, A. M., Clarimont, C. Improving the Decision-Making Process in Structural Modification of Drug Candidates: Reducing Toxicity. Drug Disc. Today. 2004, 9, 1055–1064.

23. The American Heritage Dictionary of the English Language (4th ed.). New York: Houghton Mifflin, 2000.

24. Sneader, W. Drug Prototypes and their Exploitation. New York: Wiley, 1996, pp. 469–478.

25. Sternbach, L. H. The Benzodiazepine Story. J. Med. Chem. 1979, 22, 1–7.

26. Wermuth, C. Selective Optimization of Side Activities: Another Way for Drug Discovery. J. Med. Chem. 2004, 47, 1303–1314.

27. Greenwood, D. T., Mallion, K. B., Todd, A. H., Turner, R. W. 2-Aryloxymethyl-2,3,5,6-tetrahydro-1,4-oxazines: A New Class of Antidepressants. J. Med. Chem. 1975, 18, 573–577.

28. Kling, J. From Hypertension to Angina to Viagra. Mod. Drug Disc. 1998, 1(2), 31–38.

29. Newman, D. J., Cragg, G. M. Natural Products as Sources of New Drugs over the Last 25 Years. J. Nat. Prod. 2007, 70, 461–477.

30. Quinn, R. J., Carroll, A. R., Pham, N. B., Baron, P., Palframan, M. E., Suraweera, L. Developing a Drug-Like Natural Product Library. J. Nat. Prod. 2008, 71, 464–468.

Received on 03.01.2021 Modified on 29.01.2021

Res. J. Pharmacology and Pharmacodynamics.2021; 13(2):46-50.

DOI: 10.52711/2321-5836.2021.00010