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 (pecific and ongeneticAP-dependent roteinasers) 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 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.

No abstract

No abstract

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.

The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin–proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

RESULTS Structure Table 1. (Fig. 3). Degradation of cIAP1 and XIAP by SNIPER(ER)s.As observed in studies of many IAP antagonists, SNIPERs in our study rapidly ( Fig. 2a and 3b), consistent with their increased binding affinities for cIAP1 (Table 1). Further, these SNIPER(ER)s reduced XIAP expression in MCF-7 cells after 48 h in a more potent fashion than SNIPER(ER)-87( Fig. 2a). The reduction of XIAP by SNIPER(ER)s was prominent in T47D cells after 48 h, but was weak after 4 h of exposure (Fig. 3b). The difference in the degradation pattern between cIAP1 and XIAP suggests that these IAPs are degraded through a different mechanism, as discussed below. XIAP is required for the ERα degradation by SNIPER(ER)s.We previously reported that XIAP is In vivo protein knockdown activities and antitumor activities of SNIPER(ER)sWe measured the metabolic stabilities of Development of potent SNIPER derivatives against ERα 9MCF-7 cells, but not in T47D cells (Fig. 6).The role of IAPs in cancer cell survival has been suggested in many papers (32,(42)(43)(44)(45)58 Measurement of binding affinities of ERα and IAPsThe bindings between test compounds and cIAP1, cIAP2 or XIAP were determined by

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.

No abstract