title: Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.
abstract: Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein.
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Okuhira
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Takuji
authors.initials: T
authors.lastname: Shoda
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Risa
authors.initials: R
authors.lastname: Omura
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Nobumichi
authors.initials: N
authors.lastname: Ohoka
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hattori
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Norihito
authors.initials: N
authors.lastname: Shibata
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Demizu
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Sugihara
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Asato
authors.initials: A
authors.lastname: Ichino
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Kawahara
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Itoh
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Ishikawa
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hashimoto
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kurihara
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Itoh
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
authors.affiliation: Division of Molecular Target and Gene Therapy Products (K.O., N.O., T.H., N.S., M.N.), and Division of Organic Chemistry (T.S., Y.D., M.K.), National Institutes of Health Sciences, Tokyo; Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima (K.O., R.O., R.S., A.I., H.K., H.S.); Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (Y.I., M.I., Y.H.); Showa Pharmaceutical University, Machida (S.I.), Japan miki-naito@nihs.go.jp.
authors.forename: Mikihiko
authors.initials: M
authors.lastname: Naito
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D024101:Mitochondrial Proteins / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D059748:Proteolysis; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D054852:Small Molecule Libraries / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: D024101:Mitochondrial Proteins; D011506:Proteins; D018168:Receptors, Retinoic Acid; D054852:Small Molecule Libraries; D051636:X-Linked Inhibitor of Apoptosis Protein; C082928:retinoic acid binding protein II, cellular; D046988:Proteasome Endopeptidase Complex