Identifying the Cellular Targets of Bioactive Small Molecules with Activity-Based Probes

Abstract

The renaissance of cell- or organism-based phenotypic assays has made subsequent target identification for bioactive small organic molecules an important aspect of current drug discovery. Among the many strategies available for target identification, derivatizing bioactive small molecules into activity-based probes has the main advantage of determining small molecule-protein interactions directly in the native environment where the target proteins maintain their three-dimensional structures, including all the post-translational modifications, as the discrete small molecular probes usually have better access to intracellular compartments. Thus this chemical platform will not only afford a more precise means of understanding the mechanisms of action for bioactive molecules, but shed light onto the specificity of the bioactive small molecules. Here we will provide an overview of the strategies for the design of activity-based small molecular probes and review their applications for target identification using case studies. Special emphasis is placed on logistic concerns for probe's design as well as recent developments in this field.

Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins

“…19, 20, 25 The general idea appears to have been first described over 20 years ago for the isolation of ACTH receptors, 39 and successfully implemented a few years later for isolation of a melanocyte-stimulating hormone receptor. 40 In both cases, biotin had been selected as the affinity purification agent because of its high affinity for streptavidin, which in principal would enable facile separation of a protein bearing a biotinylated ligand from non-target proteins.…”

“…19, 20 The isolated proteins are then separated and identified by bottom up mass spectrometry. There have been several reports in which compounds with K d 's of around 1 nM can be used to pull down target proteins in this manner from cell lysates.…”

“…21-23 To identify more weakly binding proteins, phage display libraries have been used in place of cell lysates to afford a higher concentration of protein, 24 although this approach is limited by the contents of the library. Another approach is to increase the affinity of the compound by attaching a photocrosslinking agent, 19, 20, 25 or by making use of intrinsic photoaffinity properties of the compound. In all cases, biotin must be attached to the compound at a position that does not interfere with target protein binding.…”

Network Pharmacology Strategies Toward Multi-Target Anticancer Therapies: From Computational Models to Experimental Design Principles

“…High-throughput experimental techniques for rapid evaluation of drug-target interactions have been developed, including cell-based phenotypic screens [45], binding affinity assays [46], in vitro modeling of ADMET (absorption, distribution, metabolism, excretion and toxicity) [47], activity-based probes [48] and transcriptional profiling [49]. However, the current screening capacities are still limited in testing the huge amount of possible compounds against hundreds of therapeutically relevant protein targets.…”

Transcript Profiling and RNA Interference as Tools To Identify Small Molecule Mechanisms and Therapeutic Potential

“…6, 7 While quantitative proteomic mass-spectrometry techniques have significantly aided the distinguishing of low-abundance protein targets from high abundance non-specific small molecule-protein interactions, 8 there remain fundamental drawbacks associated with affinity reagent based target identification strategies. Such projects are typically labor-intensive, with no guarantee of success.…”