Design And Activity Analysis Of Aptamer-based PROTAC For Targeted Degradation Of Nucleolin

The emerging PROteolysis TArgeting Chimeras(PROTACs)are heterobifunctional molecules harboring one ligand that binds to a POI and a covalently linked ligand of an E3 ubiquitin ligase,leading to the degradation of protein targets via the ubiquitin-proteasome system.PROTACs are revolutionizing conventional drug discovery modality to target proteins of interest(POIs)that were categorized as “undruggable” before,however,these strategies are still limited by the identification of suitable ligands for most proteins,especially for structural proteins.Aptamers,also termed as "chemical antibodies",are short and single-stranded nucleic acid oligonucleotides with unique three-dimensional conformations.They are typically selected by the technique of systematic evolution of ligands by exponential enrichment(SELEX).Aptamers not only have all of the advantages of antibodies,but also have unique advantages,such as small size,high stability,low cost,easy synthesis and convenient modification,low immunogenicity and wide range of targets.The unique structure of aptamers is essential for their binding to target proteins,and any protein can virtually be specifically recognized by aptamers.Based on these advantages of aptamers,we developed a novel strategy for inducible degradation of undruggable proteins by exploiting aptamers as targeting warhead.Nucleolin(NCL)is one of the most abundant proteins in the nucleolus,mainly located in the dense fibrillogenic and granular regions of the nucleolus.NCL is involved in various cellular activities such as chromatin structure maintenance,transcription,ribosome assembly,nucleoplasmic transport,and cell cycle regulation.NCL is highly expressed in diverse tumors,and it promotes the oncogenesis and progression through upregulating proliferation and migration,and inhibiting apoptosis of tumor cells,indicating that NCL might be a promising target for cancer therapy.Therefore,we selected NCL for proof-of-concept and developed a series of NCL PROTACs by exploiting aptamers as targeting warhead.Western Blotting results revealed that one of the PROTACs,d NCL#T1,displayed an effective degradation.Microscale thermophoresis(MST)results showed that d NCL#T1 had a high affinity with NCL,and further studies revealed that d NCL#T1 induced NCL degradation in ubiquitinproteasome dependent manner.And then,we analyzed the anti-tumor activity of d NCL#T1 by apoptosis assay,three-dimensional tumor culture assay,migration assay and cell colony formation assays.The results showed that d NCL#T1 could promote apoptosis and inhibit the growth,migration and proliferation of breast cancer cells.To reduce the on-target toxicity of d NCL#T1,we further developed a light-controlled PROTAC,opto-d NCL#T1,by introducing a complementary oligonucleotide(Q-CP)to hybridize with d NCL#T1.UVA irradiation broke the Q-CP complementary chain and liberated the d NCL#T1from the caged hybrid,leading to d NCL#T1 activation.Taken together,these results showed that d NCL#T1 has significant degradation effect on oncogenic protein NCL.d NCL#T1 can significantly inhibit the growth,migration and proliferation of breast cancer cells,indicating that apt-PROTAC is an effective strategy for targeting protein degradation.Further,we demonstrated that the activity of opto-d NCL#T1 can be modulated by switching the conformation of the aptamer.Thus,apt-PROTAC is an alternative and viable approach to spatiotemporally regulate the target protein.