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The HaloPROTAC utilizes the HaloTag protein, an engineered bacterial dehalogenase that allows covalent conjugation of a chloroalkane-labeled molecule to a fusion protein of interest. HaloPROTACs have been designed to induce degradation of various HaloTag fusion substrates, including cytosolic, endosomal and nuclear-localized proteins. Creative Biolabs provides a full range of products and services based on our HaloPROTAC platform. We are experienced and dedicated to helping customers in areas of PROTAC development.
HaloTag fusion proteins have been widely used as a method to bio-orthogonally label proteins in vivo. Due to HaloTag’s resistance towards alternative methods of induced degradation and the existence of a potent small-molecule ligand capable of accommodating long linkers, HaloTag fusion proteins would make an ideal model system to develop novel small molecule HaloPROTACs.
Both VHL-based and cIAP-based HaloPROTACs have been designed to induce ubiquitylation and degradation of HaloTag7 fusion proteins. In cells, these HaloPROTACs covalently bound to the HaloTag fusion protein and recruited the desired E3 ligase to facilitate ubiquitin transfer, leading to subsequent degradation by the proteasome. Researchers have demonstrated that, similar to standard PROTAC technology, the composition of the E3 ligase binder, the linker attachment point, and linker length were important factors affecting degradation efficiency. This technology was applied to degrade a range of targets, including GFP, ERK1, MEK1, TNFa, Cdc42, CREB1 and c-Jun, demonstrating the generality of the approach. As plasmids containing HaloTag fused with 20000 human genes are commercially available, this technology should be applicable to study the degradation of numerous proteins, regardless of whether known binders/inhibitors exist.
Fig.1 Schematic depiction of a bifunctional HaloPROTAC.
To expand the applications of HaloTag, the combination of HaloPROTACs with CRISPR/Cas9 genome editing was used to induce the rapid degradation of endogenous proteins. CRISPR/Cas9 genome editing has been used to insert a HaloTag at the N terminus of VPS34 and the C terminus of SGK3, whilst preserving endogenous expression levels and biological functions. These HaloTag fusion proteins were successfully degraded by a VHL-based HaloPROTAC, with degradation potency in the nmol range and high selectivity. This technique is likely to have a great impact on the study of protein function, especially of proteins for which high-affinity ligands are not yet available.
HaloPROTAC will inspire the development of future PROTACs with more drug-like properties. The HaloPROTACs are also useful chemical genetic tools, due to their ability to chemically knockdown widely used HaloTag fusion proteins in a general fashion. Furthermore, the HaloPROTAC approach, combining HaloPROTAC with CRISPR/Cas9 mediated endogenous protein tagging, provides a useful tool to interrogate an endogenous system and validate the therapeutic potential of degrading its protein target.
Based on the HaloPROTAC platform, Creative Biolabs systematically deploys a series of services to fully explore the potential of PROTAC as a new generation of small molecule drugs. If you are interested in our services, please directly contact us and consult our technical supports for more details.