key: cord-0265389-hmxd9q90
authors: Shao, Jingwei; Yan, Yuqian; Ding, Donglin; Wang, Dejie; He, Yundong; Pan, Yunqian; Yan, Wei; Kharbanda, Anupreet; Li, Hong-yu; Huang, Haojie
title: Destruction of DNA-binding proteins by programmable O’PROTAC: Oligonucleotide-based PROTAC
date: 2021-03-09
journal: bioRxiv
DOI: 10.1101/2021.03.08.434493
sha: 85975d244d7014515c83fc63530ec803793cf84c
doc_id: 265389
cord_uid: hmxd9q90

DNA-binding proteins including transcription factors (TFs) play essential roles in gene transcription and DNA replication and repair during normal organ development and pathogenesis of diseases such as cancer, cardiovascular disease and obesity, deeming to be a large repertoire of attractive therapeutic targets. However, this group of proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand binding pocket. PROteolysis-TArgeting Chimera (PROTAC) technology has been developed by engineering a bifunctional small molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through proteasome pathway. Here we report the development of Oligonucleotide-based PROTAC (O’PROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI. We demonstrate that O’PROTACs of ERG and LEF1, two highly cancer-related transcription factors selectively promote degradation of these proteins and inhibit their transcriptional activity in cancer cells. The programmable nature of O’PROTACs indicates that this approach is applicable to destruct other TFs. O’PROTACs not only can serve as a research tool, but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

A large group of DNA-binding proteins act as transcription factors (TFs) that transcriptionally activate or suppress gene expression by interacting with specific DNA sequence and transcription co-regulators. Approximately 2,000 TFs have been identified in eukaryotic cells and they are associated with numerous biological processes. Among them, approximately 294 TFs are associated with cancer development, which account for ~19% of oncogenes 1 . Therefore, targeting TFs associated with cancer development appears to be an appealing strategy for cancer treatment.

In the last decades, small molecule modulators have been developed to target nuclear receptors on the basis that this class of TFs contains a clearly defined ligandbinding pocket 2 . However, most of other TFs are difficult to target because they lack a ligand binding pocket. As the knowledge regarding the mechanisms of the assembly of transcription complexes has increased exponentially, different strategies to modulate the activity of TFs with small molecule compounds have emerged, including blocking protein/protein interactions, protein/DNA interactions, or chromatin remodeling/epigenetic reader proteins 3 . However, the development of traditional small molecules inhibiting non-ligand TFs remains very challenging, and a new targeting strategy to overcome the hurdle is highly demanded.

PROTACs are heterobifunctional small molecules composed of a POI ligand as a warhead, a linker and an E3 ligase ligand. The PROTAC molecule recruits the E3 ligase to the POI and induced the ubiquitination of the latter and further degradation by the proteasome pathway. PROTAC technology has greatly advanced during the last decade. It has been proved that PROTACs are capable of degrading a variety of proteins, including enzymes and receptors [4] [5] [6] [7] . Two PROTACs, ARV-110 and ARV-471 which are androgen receptor (AR) and estrogen receptor (ER) degraders, respectively have entered phase I clinical trials [8] [9] . PROTACs offer several advantages over small molecule inhibitors including expanding target scope, improving selectivity, reducing toxicity and evading inhibitor resistance 10 12 , which utilize haloPROTAC, dCas9-HT7 and dsDNA/CRISPR-RNA chimeras to degrade TFs. Nevertheless, this approach uses the artificially engineered dCas9-HT7 fusion protein as a mediator, which limits its potential use in clinic.

ERG transcription factor belongs to the ETS family and is involved in bone development, hematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion 13 . Importantly, it is overexpressed in approximately 50% of all human prostate cancer cases including both primary and metastatic prostate cancer due to the fusion of ERG gene with the androgen-responsive TMPRSS2 gene promoter 14 .

TMPRSS2-ERG gene fusion results in aberrant overexpression of truncated ERG, implying that increased expression of ERG is a key factor to drive prostate cancer progression 15 . Therefore, therapeutic targeting ERG is urgently needed to effectively treat prostate cancer patients. Lymphoid enhancer-binding factor 1 (LEF1) is another highly cancer-related TF. It belongs to T cell factor (TCF)/ LEF1 family. Complexed with β-catenin, LEF1 promotes the transcription of Wnt target genes 16 . LEF1 also can facilitate epithelial-mesenchymal transition (EMT) 17 . Aberrant expression of LEF1 is implicated in several cancer types and related to cancer cell proliferation, migration, and invasion 18 . Hence, LEF1 is another ideal target for cancer treatment.

In the present study, we introduce a new strategy to target TFs using O'PROTACs, in which a double-stranded oligonucleotide is incorporated as POI binding moiety in PROTAC ( Figure 1 ). We demonstrate that ERG O'PROTAC promotes proteasomal degradation of ERG protein and inhibits ERG transcriptional activity. Akin to ERG degrader, LEF1 O'PROTAC induces the degradation of LEF1.

Consequently, its target gene expression and prostate cancer cell growth was also effectively inhibited.

ERG recognizes a highly conserved DNA binding consensus sequence including the 5'-GGAA/T-3' core motif 19 . We designed a 19-mer double-stranded oligonucleotide containing the sequence of ACGGACCGGAAATCCGGTT with the ERG binding moiety underscored. As for the E3 ligase-recruiting element, we selected the widely used pomalidomide and VHL-032, which are capable of hijacking cereblon and von Hippel-Lindau (VHL) respectively. PROTAC exerts its function based on the formation of ternary complex, in which a linker plays an important role. Therefore, we designed and synthesized six phosphoramidites with different linkers in different lengths and types, three of which are linked to pomalidomide and three with VHL-032 (P1-6, Table 1 ). The phosphoramidite was attached to the 5' terminal of one DNA strand through DNA synthesizer. After annealing, we generated six O'PROTACs (OPs) for both ERG and LEF1. (Table S1 ).

The synthesis of P1-6 was illustrated in Scheme 1. 4-Fluoro-thalidomide and VHL-032 were prepared according to literature procedures [20] [21] 

To extend the utility of O'PROTACs, we turned to another transcription factor LEF1. LEF1 acts as a DNA binding subunit in the β-catenin/LEF1 complex and exerts transcriptional regulation via binding to the nucleotide sequence 5'-A/TA/TCAAAG-3' 23 . We designed 18-mer double-stranded oligonucleotide containing the sequence of TACAAAGATCAAAGGGTT as the LEF1 binding moiety. Six LEF1 O'PROTACs (Table S1) were synthesized using the same protocol as for the ERG O'PROTACs.

We first evaluated the degradation capability of each LEF1 O'PROTACs in PC-3 prostate cancer cell line. Western blot assay was utilized to detect the expression of Collectively, LEF1 OP-V1 is a potent LEF1 degrader.

In this study we take a new strategy of degrading "undruggable" transcription factors by employing O'PROTACs. O'PROTAC exploits natural "ligand" of transcription factors, namely specific DNA sequence, attached to an E3 ligase ligand via a linker. The tactic has been successfully applied to degrade ERG and LEF1 TFs with potent efficacy in cultured cells.

Conventional PROTAC technology is rapidly evolving with some of them are in clinical trials; however, it inherits certain limitations. First, most of the reported PROTACs rely on the existing small molecules as POI targeting warhead, which make it difficult to be applied to "undruggable" targets like TFs. Additionally, due to their high molecular weight (600~1400 Da), PROTACs suffer from poor cell permeability, stability and solubility 24 Hall and colleagues recently report RNA-PROTACs, which utilize singlestranded RNA (ssRNA) to recruit RNA-binding protein (RBP). The binding of RBP with RNA heavily relies on both sequence motif and secondary structure 25-26 .

Predicting the interaction between RNA and RBP is challenging due to the high flexibility of RNA [27] [28] . However, double-stranded DNA bear a well-defined threedimensional duplex structure; therefore, the protein binding region is accessible and predictable. Hence, O'PROTAC is programmable by changing the nucleotide sequence that binds protein. Additionally, compared with double-stranded oligonucleotide, ssRNA is susceptible to deleterious chemical or enzymatic attacks 28 .

Taken together, O'PROTAC is desirable due to its readily predictability and superior stability.

Oligonucleotide drug development has become a main stream for new drug hunting in the last decade 29 . The catalytic advantage of PROTACs 30 incorporated into oligonucleotide drugs could further fuel the field. Moreover, the delivery of oligonucleotide drugs has been advanced significantly in the recent years, notably for mRNA COVID-19 vaccine [31] [32] . Therefore, O'PROTACs can be a complementary drug discovery and development platform to conventional PROTACs to derive clinical candidates and accelerate drug discovery. PLAT and PLAU). P values were calculated using the unpaired two-tailed Student's ttest; * P < 0.05; ** P < 0.01; *** P < 0.001, n.s., not significant. 

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