Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

Abstract

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation, identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed.

Fig. 1

Typical breakthrough curve obtained in a FAC assay.

Fig. 2

Representative illustrations of FAC direct assays for binders screening and characterization. In ranking experiments (A), a compounds mixture is continuously infused at a known concentration onto the bioaffinity column: the breakthrough curve 1 represents the elution of a non-affinity compound; the breakthrough curve 2 represents the elution of a compound with moderate affinity to the immobilized target; the breakthrough curve 3 represents the elution of the most potent binder presents in the evaluated mixture. The dissociation constant (K_d) can be accurately obtained by the infusion of individual concentrations of the binder (B), or in a single assay with the sequential infusion of the binder at increasing concentrations by the modified staircase method (C).

Fig. 3

In displacement studies, the breakthrough curve of a marker (I) is monitored. When the analyzed solution contains the marker and an evaluated compound (or a compounds mixture), the breakthrough curve of the marker could be displaced (II), suggesting a direct competition between the binder and the evaluated compound (or at least one compound in a mixture) competes directly for a specific binding site.

Fig. 4

Block diagram of 2DLC system. (A) Position 1 and (B) position 2.

Fig. 5

(A) Schematic workflow for some general ligand fishing assays. (B) Scheme for the ligand fishing assay with magnetic beads and (C) with hollow fibers.