**3. Affinity chromatography and drug discovery**

Multidentate chelating compounds are widely used in order to strengthen the complex which is comprised of chelator, metal ion and protein. Different length of spacers is used to bind the chelator onto the surface of the support. Type of the chelator influences the strength of the chelation and retention power, for instance metal binds to the nitrogen atom and two carbox‐ ylate oxygens and reveals three free sites in case of tridentate iminodiacetic acid (IDA); tetradentate nitrilotriacetic acid (NTA) binds the metal by an additional carboxylate oxygen and this provides stronger chelation, but a weaker retention power. IDA is the chelator which is commonly used in the applications of IMAC. Although most of the chelators are carboxy‐ methylated amines, there also some other compounds which are commonly used such as dyeresistant yellow 2KT, OPS and 8-HQ [26]. Some examples of chelating compounds are given

In case of use boronic acid or boronates as ligand of the affinity chromatography, this type of methods are called boronate affinity chromatography. Most of the boronate derivatives are known to bind compounds with cis-diol groups covalently at a pH above 8. Separation of glycoproteins from non glucoprotein structures is possible by boronate affinity method due to cis-diol groups of the sugars. For instance, this method may successfully performed to seperate glucohemoglabin and normal hemoglobin or to determine different types of glyco‐

There are many other chromatographic methods which are closely related to traditional affinity chromatography. For example **Analytical Affinity Chromatography** (**Quantitative Affinity Chromatography** or **Biointeraction Chromatography**) which is used as a tool for determination of solute-ligand interactions [21]. It is possible to investigate several biological systems, such as lectin/sugar, enzyme/inhibitor, protein/protein, DNA/protein interactions as well as binding of drugs or hormones to serum proteins with this technique. Thus competition of drugs with other drugs or endogenous compounds for protein binding sites may success‐ fully be detected by this method. Either immobilized drugs or immobilized proteins may be used in the studies about drug-protein and hormone-protein binding, although protein-based columns which may be use for multiple experiments are more common [1,29]. The competition between two solutes for binding sites can also be examined by this method and this technique is known as **Frontal Affinity Chromatography** [21]. **Hydrophobic Interaction Chromatogra‐ phy** and **Thiophilic Adsorption** methods also related to affinity chromatography. Immobi‐ lized thiol groups are used as ligands in **Thiophilic Adsorption** (**Covalent/Chemisorption Chromatography**) in order to separation of sulfhydryl-containing peptides or proteins and mercurated polynucleotides. In **Hydrophobic Interaction Chromatography** short non-polar chain, such as those that were originally used as spacer arms on affinity supports provide binding with proteins, peptides and nucleic acids. **Chiral Liquid Chromatography** methods can be also considered as affinity based techniques [21]. These techniques are widely utilized in pharmaceutical industry and clinical chemistry for the separation of individual chiral forms of the drugs and the quantification of different chiral forms of drugs or their metabolites. Since most of the ligands used in affinity chromatography are chiral, they may be preffered as

in Table 5.

92 Column Chromatography

**2.6. Boronate affinity chromatography**

proteins in a sample [1].

There are a number of areas related to affinity chromatography that have also been of great interest in pharmaceutical and biomedical analysis. One such area is the use of affinity chromatography in drug discovery [12]. In drug discovery explaining the mechanism of action of bioactive compounds, which are used as pharmaceutical drugs and biologically active natural products, in the cells and the living body is important. For this pupose isolation and identification of target protein(s) for the bioactive compound are essential in understanding its function fully. Affinity chromatography is a useful method capable of isolating and identifying target molecules for a specific ligand, utilizing affinity between biomolecules such as antigen–antibody reactions, DNA hybridization, and enzyme–substrate interactions. Since the development of affinity chromatography in the early 1950s, various types of target proteins for bioactive compounds have been isolated and identified. Selected samples of the target proteins isolated by affinity chromatography are listed in Table 6. Since then, affinity chro‐ matography has been gaining renewed attention as a widely applicable technique for the discovery the target proteins for bioactive compounds [30].

phy are commonly used types of these techniques. Affinity chromatography which provides analysis of bioactive molecules based on their biological functions or individual struc‐ tures has become increasingly important as another liquid chromatography technique [1,12]. Affinity chromatography is based on the simple principle that every biomolecule recog‐ nize another natural or artificial molecule such as enzyme and substrate or antibody and antigen [2,7]. In fact this technique is one of the oldest forms of liquid chromatography method [8]. The first use may be considered as the isolation of α-amylase by using an insoluble substrate, starch, in 1910 just three years after the discovery of chromatography by Tsewett [6,9]. The modern applications have started since 1960s with the creation of beaded agarose supports and the use of cyanogens bromide immobilization method. Since then, affinity chromatography has been gaining attention as a widely applicable techni‐ que for discovering the target proteins for bioactive compounds [3]. Up to the present time many different types of target proteins for bioactive compounds have been isolated and identified by affinity chromatography [21]. Today affinity chromatography is utilized as a valuable technique for the separation, purification and analysis of compounds present in complex samples and used in biochemistry, pharmaceutical science, clinical chemistry and environmental sciences. Application of affinity chromatography has significant advantag‐ es. The important one is that affinity chromatography involves many types of interac‐ tions between ligand and target such as steric effects, hydrogen bonding, ionic interactions, van der Waals forces, dipol-dipol interactions and even covalent bonds while other chromatographic techniques involve just one or a few of them. The combination of these multiple interactions leads to separation with high selectivity and retention in affinity chromatography [8]. However there are also several drawbacks in affinity chromatogra‐ phy system, such as non-specific binding of irrelevant proteins during affinity purifica‐ tion and chemical modification of bioactive compounds of interest used as ligands. These drawbacks limit its extensive application. After the completion of the Human Genome Project drug discovery research has focused on an approach that includes identification and characterization of molecular and cellular functions of a wide variety of proteins encoded by genomes. Thus, bioactive compounds become more important not only as therapeutic agents to treat diseases and disorders but also as useful chemical tools to examine their complex biological processes in vitro and in vivo [30]. Affinity chromatography which allows explaining the mechanism of action of bioactive compounds that are used as pharmaceutical drugs and biologically active natural products, therefore, has also signifi‐

Affinity Chromatography and Importance in Drug Discovery

http://dx.doi.org/10.5772/55781

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cant importance in modern drug discovery [30].

Özlem Bahadir Acikara, Gülçin Saltan Çitoğlu, Serkan Özbilgin and Burçin Ergene

Ankara University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey

**Author details**

**Table 6.** Selected examples of the target protein(s) isolated and identified through affinity chromatography [30]
