Pharmacological difference between degrader and inhibitor against oncogenic BCR-ABL kinase.

Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.

Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation.

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 receptor α (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.

Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands.

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.

In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs).

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

SNIPER(TACC3) induces cytoplasmic vacuolization and sensitizes cancer cells to Bortezomib.

We previously developed a hybrid small molecule SNIPER (Specific and Nongenetic IAP-dependent Protein ERaser) against transforming acidic coiled-coil-3 (TACC3), SNIPER(TACC3), that induces proteasomal degradation of TACC3 protein. In this study, we found that SNIPER(TACC3) induces cytoplasmic vacuolization derived from endoplasmic reticulum (ER) and paraptosis-like cell death selectively in cancer cells. Mechanistic analysis suggests that accumulation of ubiquitylated protein aggregates that requires X-linked inhibitor of apoptosis protein (XIAP) induces ER stress, which results in ER-stress responses involving X-box binding protein-1 (XBP-1) and ER-derived vacuolization in cancer cells. Importantly, inhibition of proteasome enhanced the SNIPER(TACC3)-induced vacuolization, and the combination treatment of SNIPER(TACC3) and bortezomib exhibited a synergistic anticancer activity in several cancer cell lines. The induction of paraptosis-like cell death in cancer cells by SNIPER(TACC3) could be applied to treat cancer cells resistant to undergo apoptosis by overexpression of XIAP.