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

Abstract

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.

Targeted Protein Degradation: from Chemical Biology to Drug Discovery

“…Thus far, SNIPERs have successfully degraded multiple cellular targets, e.g., Cellular Retinoic Acid-Binding Protein I (CRABPI) and CRABPII (Itoh et al, 2010; Itoh et al, 2012; Okuhira et al, 2017), ERα (Itoh et al, 2011b; Demizu et al, 2012; Okuhira et al, 2013), the spindle regulatory protein Transforming Acidic Coiled-Coil-3 (TACC3) (Ohoka et al, 2014), the Breakpoint Cluster Region-Abelson tyrosine kinase (BCR-ABL) (Demizu et al, 2016) and various HaloTag fusion proteins (Tomoshige et al, 2016). Recently, the introduction of the IAP antagonist LCL161 has boosted SNIPER activity to the low nanomolar range, and allowed SNIPER evaluation in mouse xenograft models for the first time (Ohoka et al, 2017). Nevertheless, simultaneous IAP degradation and incomplete target degradation remain issues of this technology and further studies are necessary to evaluate its therapeutic potential.…”

The PROTAC technology in drug development

“…45 The designed PROTAC named MZ1 dramatically induced degradation of BRD4. [58][59][60][61][62][63][64][65] Unexpectedly, they deciphered that one of their PROTAC based on bestatin did not recruit cIAP-1 but instead APC/C CDH1 complex. 49 Inhibitors including imatinib, bosutinib, and dasatinib were linked to VHL E3 ubiquitin ligase ligand or pomalidomide (to recruit cereblon E3 ubiquitin ligase).…”

Chemical Protein Degradation Approach and its Application to Epigenetic Targets

“…Inspired by these findings, SNIPERs have been widely applied to target several proteins, including nuclear receptors and huntingtin protein ( 8 and 9 , Figure ) . Naito's group is vigorously advancing SNIPERs and has reported that an ER SNIPER ( 10 , Figure ), designed based on the ER ligand 4‐hydroxy‐tamoxifen and the IAP ligand, LCL161, reduces ER levels in tumor xenografts and suppresses the growth of ER‐positive breast tumors in mice . Naito's group also developed a SNIPER targeting bromodomain proteins (see section 3.1) …”

“…Structural optimization of BET PROTACs is currently underway. Optimization of a linker, such as PEG, methylene, or triazole‐constructed by click chemistry and the combination of BET inhibitors with E3 ligase binders, including an IAP ligand, have been reported (compounds 35–39 , Figure ) ,. In addition, chemical biology studies using BET PROTACs have been performed ,,.…”