Target deconvolution techniques in modern phenotypic profiling

Abstract

The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called 'target deconvolution', is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in 'omics' technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of 'modern' phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.

Figure 1

Affinity Chromatography. (A) An active hit is directly linked to a solid support or tagging group such as biotin, and its target protein isolated via affinity pull-down. The isolated proteins are further analyzed by mass spectrometry; (B) An active hit is modified with a small ‘clickable’ group for in situ labeling with minimal structural perturbation; (C) A photo-reactive group such as diazirine group is added to induce covalent cross-link between the hit compound and its target.

Figure 2

Activity-Based Protein Profiling. (A) Three main features of activity-based probes: 1) A reactive electrophile that allows covalent attachment of the ABP in the active site of target enzyme, and enables rapid isolation and analysis of the target protein, 2) A specificity region that directs the probe to the specific class of enzymes, and 3) A tagging group for detection; (B) General workflow for using an ABP. When an active hit is identified from a screen it can be used to directly affinity isolate the target (top). Similarly, the direct screening of ABPs and covalent inhibitors in various systems such as cells and animals greatly facilitates the overall process of target deconvolution. Furthermore, broad-spectrum ABPs provide a powerful tool to study disease related pathways and class-wide enzyme assay platform.

Figure 3

Label-free techniques for target deconvolution. (A) Limited proteolysis techniques such as DARTS and pulse proteolysis utilize stability of protein-ligand complex under proteolytic condition. Ligand bound proteins are more resistant to proteolysis in the presence of denaturant (pulse-proteolysis) or without denaturant (DARTS), and non-binding proteins are hydrolyzed to small peptides and amino acids. All proteolysis resistant proteins can be analyzed by SDS-PAGE and identified by mass spectrometry; (B) SPROX technique is based on a similar principle, however, it exploits protein-ligand interaction under oxidative conditions in various concentrations of denaturant. Ligand bound proteins are more resistant to oxidant, thus require higher amounts of denaturant to generate the same degree of oxidation compared to non-binders. These results can be plotted and protein-ligand interactions result in a right shift of the plot.

Figure 4

Expression cloning techniques. Proteins can be expressed using cloning vectors containing cDNA library, and these proteins exposed to small molecules for affinity selection. (A) Phage display. Small molecule captured phage particles can be selectively eluted and transfected into bacterial cells for further amplification and enrichment; (B) mRNA display. mRNA display utilizes an in vitro translation system to generate a library of mRNA-protein fusions, and this newly generated library can be exposed to an immobilized small molecule. After affinity selection, the cDNA of the captured proteins can be amplified by PCR and used to identify the target or for a next round of selection for further enrichment; (C) Yeast three hybrid screen. Screen constructs consists of a bait domain containing a DNA binding domain fused to a protein of interest, and a prey domain containing a transcriptional activator for a reporter gene linked to a library of proteins encoded by a cDNA library. When the bait and prey domains interact through the small molecule dimerizer, transcription of a reporter gene is activated.