Targeted degradation of proteins localized in subcellular compartments by hybrid small molecules

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 Non-genetic 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 SNIPERs are able to induce degradation of 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)s with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1, induce the proteasomal degradation of wild-type, cytosolic, nuclear and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity towards the mitochondrial CRABP-II protein. The siRNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP)s against wild-type 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 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.