title: Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.
abstract: We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins.
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Shusuke
authors.initials: S
authors.lastname: Tomoshige
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hashimoto
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address: m-ishikawa@iam.u-tokyo.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Ishikawa
mesh_terms: D066241:Carbon-13 Magnetic Resonance Spectroscopy; D057809:HEK293 Cells; D006801:Humans; D066244:Proton Magnetic Resonance Spectroscopy; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors*; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020764:cdc42 GTP-Binding Protein
keywords: HaloTag; Protein knockdown; Ubiquitin ligase