Labelled Chemical Probes for Demonstrating Direct Target Engagement in Living Systems

Abstract

Demonstrating target engagement in living systems can help drive successful drug discovery. Target engagement and occupancy studies in cells confirm direct binding of a ligand to its intended target protein and provide the binding affinity. Combined with biomarkers to measure the functional consequences of target engagement, these experiments can increase confidence in the relationship between in vitro pharmacology and observed biological effects. In this review, we focus on chemically and radioactively labelled probes as key reagents for performing such experiments. Using recent examples, we examine how the labelled probes have been employed in combination with unlabelled ligands to quantify target engagement in cells and in animals. Finally, we consider future developments of this emerging methodology.

Development of an 18F-Labeled Irreversible Inhibitor of Transglutaminase 2 as Radiometric Tool for Quantitative Expression Profiling in Cells and Tissues

“…29 Furthermore, as the transamidase function can be induced in the presence of Ca 2+ , such probes would enable the convenient and quantitative measurement of TGase 2 expression levels in vitro. In addition, activity-based probes potentially enable the assessment of target occupancy under therapeutic inhibitor treatment, 37,38 which would support drug discovery for TGase 2.…”

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors

“…To measure PCSK9, a magnetic bead-based affinity capture method for the separation of free, unbound PCSK9 from inhibitor-bound PCSK9 in plasma samples was developed as the basis for a direct target engagement assay. , Given the high affinity binding of our lead peptide series for PCSK9, we proceeded with direct biotinylation of the primary amino functional group of peptide 50 to prepare biotinylated bait peptide 51 for the bead capture method. , Free PCSK9 bound to the bait peptide loaded onto the beads in the assay is dissociated from beads and trypsinized, and a selected tryptic peptide is quantified by LC–MS/MS (details are provided in the Experimental Section).…”

Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag

“…Such optimization often requires significant synthetic effort and is difficult in the absence of structural and binding information. 1,2 Not unexpectedly, incorporation of diazirine in a region of the compound that permits retention of bioactivity and is compatible with CuAAC ligation such as the solvent exposed region could lead to rapid carbene quenching or enhanced nonspecific cross-linking 2,11 Here, we introduce a simplified enrichment strategy utilizing a single "three-in-one" photoreactive cleavable chloroalkane capture tag. This tag couples capacity for photo-crosslinking with traits of previously described chloroalkane capture tags.…”

“…Traditional enrichment methods utilize bioactive compounds tethered to either a capture tag or a solid support to facilitate selective isolation of their protein targets, often from cellular lysates. ,− ,, These enrichment methods are frequently constrained by difficulties to preserve transient binding interactions, particularly those of low to moderate affinity and/or fast dissociation rates. ,,− Furthermore, it is generally accepted that physiological elements influencing target engagement in intact cells cannot be accurately preserved in cellular lysates. Cell lysis disrupts critical elements such as protein complexes, concentrations of competing metabolites, and highly regulated subcellular localization of protein pools. ,,− It can also compromise the structural integrity of some targets, such as membrane proteins. ,, …”

“…The two small groups can be incorporated at separate sites of a compound or introduced as a single integrated unit, minimizing perturbation to a bioactive compound. , Intuitively, the propensity of diazirine’s highly reactive intermediate (i.e., carbene) for rapid quenching by water , necessitates incorporation of diazirine to the tethered compound such that it is positioned within the binding pocket of interacting targets. Such optimization often requires significant synthetic effort and is difficult in the absence of structural and binding information. , Not unexpectedly, incorporation of diazirine in a region of the compound that permits retention of bioactivity and is compatible with CuAAC ligation such as the solvent exposed region could lead to rapid carbene quenching or enhanced nonspecific cross-linking , …”