Abstract
Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.
Highlights
The use of solid-supported proteins to evaluate protein-protein interactions and to purify proteins is a well-founded tool (Muronetz et al, 2001; Perret and Boschetti, 2018)
In the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others
Various chromatographic terms have been used. Terms such as weak affinity chromatography (WAC) (Meiby et al, 2013; Singh P. et al, 2017; Ohlson and Duong-Thi, 2018; Lecas et al, 2019), high-performance affinity chromatography (HPAC) (Hage, 2017; Li Z. et al, 2017; Beeram et al, 2018; Zhang C. et al, 2018), high-performance liquid affinity chromatography (HPLAC) (Zheng et al, 2014), biointeraction chromatography (Wainer, 2004), affinity monolith chromatography (AMC) (Lecas et al, 2019), and cellular chromatography (CC) (Ciesla et al, 2016; Xu et al, 2019) have been largely and correctly employed, but all of them could be well-settled in the general term bioaffinity chromatography, which has been introduced to differentiate them from the classic affinity chromatography
Summary
The use of solid-supported proteins to evaluate protein-protein interactions and to purify proteins is a well-founded tool (Muronetz et al, 2001; Perret and Boschetti, 2018). In the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others.
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