Specific degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein

AbstractManipulation of protein stability with small molecules is a challenge in the field of drug discovery. Here we show that cellular retinoic acid binding protein-II (CRABP-II) can be specifically degraded by a novel compound, SNIPER-4, consisting of (−)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester and all-trans retinoic acid that are ligands for cellular inhibitor of apoptosis protein 1 (cIAP1) and CRABP-II, respectively. Mechanistic analysis revealed that SNIPER-4 induces cIAP1-mediated ubiquitylation of CRABP-II, resulting in the proteasomal degradation. The protein knockdown strategy employing the structure of SNIPER-4 could be applicable to other target proteins

Development of potent SNIPER derivatives against ERα

Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptorα (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein knockdown and cytocidal activities against cancer cells requiring IAPs for survival

C/EBP homologous protein (CHOP) up-regulates IL-6 transcription by trapping negative regulating NF-IL6 isoform

AbstractInterleukin-6 (IL-6) production is up-regulated by several stimuli through the activation of transcription factors. We have previously demonstrated that CCAAT/enhancer binding protein homologous protein (CHOP) positively regulates IL-6 production at the transcriptional level in the human melanoma cell line A375. In this study, we provide evidence that CHOP up-regulates the IL-6 transcription without binding to the IL-6 promoter. CHOP dimerized more preferentially with an inhibitory isoform of nuclear factor for IL-6 expression (LIP (liver-enriched inhibitory protein)) than with a positively acting isoform (LAP, liver-enriched activator protein). These results indicate that CHOP plays an important role in IL-6 production without binding to its promoter, probably by trapping protein(s) such as LIP, which would otherwise inhibit IL-6 transcription

Development of chimeric molecules that degrade the estrogen recep-tor using decoy oligonucleotide ligands

Targeted protein degradation using chimeric small molecules, such as proteolysis-targeting chimeras (PROTACs) and specif-ic and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), has attracted attention as a method to degrade intracellular target proteins via the ubiquitin-proteasome system (UPS). These chimeric molecules target a variety of proteins using small molecules that can bind to the proteins. However, it is difficult to develop such degraders in the absence of suitable small molecule ligands for the target proteins, such as for transcription factors (TFs). Therefore, we constructed the chimeric molecule LCL-ER(dec), which consists of a decoy oligonucleotide that can bind to the estrogen receptor alpha (ERalpha) and an IAP ligand, LCL161 (LCL), in a click reaction. LCL-ER(dec) was found to selectively degrade ERalpha via the UPS. These findings will be applicable to the development of other oligonucleotide-type degraders that target different TFs

Structural Optimization of Decoy Oligonucleotide-Based PROTAC That Degrades the Estrogen Receptor

Proteolysis-targeting chimeras (PROTACs) have attracted attention as a chemical method of protein knockdown via the ubiquitin–proteasome system. Some oligonucleotide-based PROTACs have recently been developed for disease-related proteins that do not have optimal small-molecule ligands such as transcription factors. We have previously developed the PROTAC LCL-ER­(dec), which uses a decoy oligonucleotide as a target ligand for estrogen receptor α (ERα) as a model transcription factor. However, LCL-ER­(dec) has a low intracellular stability because it comprises natural double-stranded DNA sequences. In the present study, we developed PROTACs containing chemically modified decoys to address this issue. Specifically, we introduced phosphorothioate modifications and hairpin structures into LCL-ER­(dec). Among the newly designed PROTACs, LCL-ER­(dec)-H46, with a T4 loop structure at the end of the decoy, showed long-term ERα degradation activity while acquiring enzyme tolerance. These findings suggest that the introduction of hairpin structures is a useful modification of oligonucleotides in decoy oligonucleotide-based PROTACs

A Novel Regulatory Mechanism of the Bone Morphogenetic Protein (BMP) Signaling Pathway Involving the Carboxyl-Terminal Tail Domain of BMP Type II Receptor▿

Bone morphogenetic protein (BMP) signaling regulates many different biological processes, including cell growth, differentiation, and embryogenesis. BMPs bind to heterogeneous complexes of transmembrane serine/threonine (Ser/Thr) kinase receptors known as the BMP type I and II receptors (BMPRI and BMPRII). BMPRII phosphorylates and activates the BMPRI kinase, which in turn activates the Smad proteins. The cytoplasmic region of BMPRII contains a “tail” domain (BMPRII-TD) with no enzymatic activity or known regulatory function. The discovery of mutations associated with idiopathic pulmonary artery hypertension mapping to BMPRII-TD underscores its importance. Here, we report that Tribbles-like protein 3 (Trb3) is a novel BMPRII-TD-interacting protein. Upon BMP stimulation, Trb3 dissociates from BMPRII-TD and triggers degradation of Smad ubiquitin regulatory factor 1 (Smurf1), which results in the stabilization of BMP receptor-regulated Smads and potentiation of the Smad pathway. Downregulation of Trb3 inhibits BMP-mediated cellular responses, including osteoblast differentiation of C2C12 cells and maintenance of the smooth muscle phenotype of pulmonary artery smooth muscle cells. Thus, Trb3 is a critical component of a novel mechanism for regulation of the BMP pathway by BMPRII