In Vivo Pretargeted Imaging of HER2 and TAG-72 Expression Using the HaloTag Enzyme

A novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands ¹¹¹In-HTL-1, -2, and -3 were evaluated as secondary agents. While this approach was not sufficiently sensitive to detect the internalizing HER2 antigen, pretargeting experiments involving the most optimal secondary agent, ¹¹¹In-HTL-3, were successful in detecting the noninternalizing antigen TAG-72 and provided high-contrast SPECT images at 4 and 24 h postinjection

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington’s disease

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag

The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and “off-targets” exhibiting a range of binding affinities, cellular abundances, and binding characteristics

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington’s disease