Affinity-Based Methods in Drug-Target Discovery

Abstract

Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

How Physiologic Targets Can Be Distinguished from Drug-Binding Proteins

“…Biochemical/chemical approaches, judiciously used, offer a direct path to finding drug-binding proteins, because the assays (e.g., photo-affinity labeling) track physical interactions between drug and protein targets. Column affinity chromatography in which drugs are coupled to matrices have been used to identify drug-binding proteins (Jones et al, 2015;Katiyar et al, 2013;Mercer et al, 2011;Rylova et al, 2015;Schenone et al, 2013;Shi et al, 2012;Thomas et al, 2017). Here it is important to remember that proteins eluted from the column, even after rigid washing protocols (Amarasinghe and Jin, 2015;Mensa-Wilmot et al, 1995;Mishra, 2020), might not bind the drug that is coupled to the chromatography matrix; some eluted proteins may be present on the column due to interaction with other proteins that bound directly to the drug.…”

Targeting the Oncogenic TBX2 Transcription Factor With Chromomycins

“…These involve the use of a molecular probe that is specifically designed to identify one or more target proteins from many others present in a complex cell lysate mixture. The identification and quantification of the isolated targets are ascertained by liquid-chromatography coupled to tandem mass spectrometry (LC-MS) (Rylova et al, 2015). Using the reverse of this approach, Lau et al (2015) screened natural crude extracts for compounds with affinity for a specific biological protein that had previously been characterized and, hence, proposed as an important therapeutic target.…”

Synthesis of imatinib, a tyrosine kinase inhibitor, labeled with carbon‐14

“…Thus, the analysis of the interactions between small molecular ligands and their molecular targets is important in innovative drug discovery and the prediction of drug toxicity . The importance of analyzing small molecule‐protein interactions has driven various target identification methods, including affinity‐based methods and proteomic target identification . Affinity‐based target identification methods, such as affinity chromatography using biotin or isotope‐tagged small molecules as affinity reagents and SILAC (Stable Isotope Labeling of Amino acids in Cell culture), have a clear disadvantage; they require the chemical modification of small molecules, which may induce responses different from those of the hit or lead compound.…”

“…analyzing small molecule-protein interactions has driven various target identification methods, including affinitybased methods and proteomic target identification. [14][15][16][17] Affinity-based target identification methods, such as affinity chromatography using biotin or isotope-tagged small molecules as affinity reagents and SILAC (Stable Isotope Labeling of Amino acids in Cell culture), have a clear disadvantage; they require the chemical modification of small molecules, which may induce responses different from those of the hit or lead compound. 14 Clabeled small molecules could therefore be an ideal choice to identify and analyze binding proteins as 14 Clabeling does not require any chemical modification.…”