Therapeutic targeting of RNA-binding protein by RNA-PROTAC
Authors: Xinyi Li, Wenchen Pu, Song Chen, Yong Peng
Journal: Molecular Therapy (2021)

Proteolysis-targeting chimeras (PROTACs) are engineered molecules that trigger the degradation of pathogenic proteins, representing a promising approach for the targeted therapy of human diseases. RNA-binding proteins (RBPs) play central roles in biological processes, and RBP dysregulation is involved in various diseases. However, RBPs are usually undruggable by conventional therapies as well as by traditional PROTACs. Recently, Ghidini et al.1Ghidini A. Cléry A. Halloy F. Allain F.H.T. Hall J. RNA-PROTACs: degraders of RNA-binding proteins.Angew. Chem. Int. Ed. Engl. 2021; 60: 3163-3169Crossref PubMed Scopus (29) Google Scholar described a new type of PROTAC, termed RNA-PROTAC, that selectively degrades RBPs, thereby expanding the PROTAC strategy for disease treatment. PROTACs induce the degradation of target proteins via the ubiquitin-proteasome system (UPS). The UPS is a highly conserved intracellular protein degradation system that degrades more than 80% of proteins in eukaryotic cells. Ubiquitin-activating enzyme E1 transfers ubiquitin to ubiquitin-conjugating enzyme E2, which subsequently recruits ubiquitin ligase E3 to drive protein ubiquitination, thereby facilitating the degradation of polyubiquitination-tagged protein by the proteasome.2Harrigan J.A. Jacq X. Martin N.M. Jackson S.P. Deubiquitylating enzymes and drug discovery: emerging opportunities.Nat. Rev. Drug Discov. 2018; 17: 57-78Crossref PubMed Scopus (324) Google Scholar Inspired by the UPS, bifunctional molecular PROTACs have been developed that comprise a ligand that binds E3 ligase, a ligand that recognizes a protein of interest (POI) and a linker between them. In cells, PROTACs hijack the UPS to drive ubiquitination and subsequent proteasome-mediated degradation of the POI, thereby achieving highly selective and effective reduction in POI abundance (Figure 1A).3Burslem G.M. Crews C.M. Proteolysis-targeting chimeras as therapeutics and tools for biological discovery.Cell. 2020; 181: 102-114Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar Unlike traditional pharmacological strategies that focus on occupying a binding site to control POI function ("occupancy-driven"), PROTACs act as catalysts to mediate protein degradation in an ongoing fashion ("event-driven").4Lai A.C. Crews C.M. Induced protein degradation: an emerging drug discovery paradigm.Nat. Rev. Drug Discov. 2017; 16: 101-114Crossref PubMed Scopus (622) Google Scholar PROTACs do not require high target affinity, high drug dosage/exposures, or targets harboring tractable active or allosteric sites, thereby minimizing the risk of undesirable off-target effects and presenting opportunities for degrading "undruggable" proteins previously considered inaccessible to small-molecule intervention.5Schapira M. Calabrese M.F. Bullock A.N. Crews C.M. Targeted protein degradation: expanding the toolbox.Nat. Rev. Drug Discov. 2019; 18: 949-963Crossref PubMed Scopus (289) Google Scholar. Existing PROTACs have been either peptide- or small molecule-based. Peptide-based PROTACs, containing a peptidic POI ligand that mimics the sequence of a native POI-binding protein, tend to be superior with regards to target specificity, binding affinity, and chemical synthesis. However, they suffer from intolerance to digestion and limited membrane permeability, rendering oral administration problematic.6Jin J. Wu Y. Chen J. Shen Y. Zhang L. Zhang H. Chen L. Yuan H. Chen H. Zhang W. Luan X. The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination.Theranostics. 2020; 10: 10141-10153Crossref PubMed Scopus (23) Google Scholar Therefore, peptide-based PROTACs are usually administered via intravenous injection and are particularly suited to the treatment of hematological diseases linked to a specific target. For example, STAT3 is a therapeutic target in leukemia and lymphoma, but most non-peptidic inhibitors exhibit low specificity for STAT3. Bai et al.7Bai L. Zhou H. Xu R. Zhao Y. Chinnaswamy K. McEachern D. Chen J. Yang C.Y. Liu Z. Wang M. et al.A potent and selective small-molecule degrader of STAT3 achieves complete tumor regression in vivo.Cancer Cell. 2019; 36: 498-511.e17Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar developed a STAT3 PROTAC (SD-36) comprising a peptide derived from the gp130 protein sequence, which binds STAT3, and a ligand for the E3 ligase CRBN. SD-36 drove selective STAT3 degradation and cured leukemia and lymphoma in vitro and in multiple xenograft mouse models (Figure 1B). Small molecule-based PROTACs, employing a small-molecule inhibitor as the POI ligand, exhibit improved resistance to digestion and increased cellular permeability compared to peptide-based PROTACs, which enables them to be administered by various routes, thereby expanding the application of PROTACs in human diseases.8Posternak G. Tang X. Maisonneuve P. Jin T. Lavoie H. Daou S. Orlicky S. Goullet de Rugy T. Caldwell L. Chan K. et al.Functional characterization of a PROTAC directed against BRAF mutant V600E.Nat. Chem. Biol. 2020; 16: 1170-1178Crossref PubMed Scopus (36) Google Scholar For example, the mutation BRAFV600E is prevalent in diverse cancers such as melanoma and colorectal cancer. Posternak et al.8Posternak G. Tang X. Maisonneuve P. Jin T. Lavoie H. Daou S. Orlicky S. Goullet de Rugy T. Caldwell L. Chan K. et al.Functional characterization of a PROTAC directed against BRAF mutant V600E.Nat. Chem. Biol. 2020; 16: 1170-1178Crossref PubMed Scopus (36) Google Scholar synthesized a small molecule-based PROTAC called P4B, comprising a POI ligand based on the small-molecule BRAFV600E inhibitor BI882370 and an E3 ligand for CRBN, which displayed effective BRAF degradation and inhibited the growth of tumor cell lines harboring BRAF mutations (Figure 1B). Notably, small-molecule ligands usually exhibit lower target specificity than peptide ligands,6Jin J. Wu Y. Chen J. Shen Y. Zhang L. Zhang H. Chen L. Yuan H. Chen H. Zhang W. Luan X. The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination.Theranostics. 2020; 10: 10141-10153Crossref PubMed Scopus (23) Google Scholar so greater consideration should be given to the specificity when selecting or optimizing small-molecule POI ligands. Despite these promising results reported for peptide- and small molecule-based PROTACs, there remain difficulties in targeting many "undruggable" proteins such as RBPs. RBPs play a central role in a wide variety of biological processes by physically associating with RNA to form RNA-protein-based machineries in cells. Aberrant expression and/or function of RBPs has been demonstrated in many diseases ranging from neurologic disorders to cancers.9Gerstberger S. Hafner M. Tuschl T. A census of human RNA-binding proteins.Nat. Rev. Genet. 2014; 15: 829-845Crossref PubMed Scopus (984) Google Scholar For instance, the overexpression of RBP Lin28 predicts poor clinical prognosis in multiple tumor types, and Lin28 inhibits the biogenesis of tumor suppressor miRNA let-7 through a direct interaction with precursor let-7 (pre-let-7), identifying Lin28 as a potential target for cancer therapy.10Viswanathan S.R. Powers J.T. Einhorn W. Hoshida Y. Ng T.L. Toffanin S. O'Sullivan M. Lu J. Phillips L.A. Lockhart V.L. et al.Lin28 promotes transformation and is associated with advanced human malignancies.Nat. Genet. 2009; 41: 843-848Crossref PubMed Scopus (642) Google Scholar,11Loughlin F.E. Gebert L.F. Towbin H. Brunschweiger A. Hall J. Allain F.H. Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28.Nat. Struct. Mol. Biol. 2011; 19: 84-89Crossref PubMed Scopus (94) Google Scholar However, the discovery of effective RBP-targeted drugs remains challenging due to RBP's structurally variable binding-pockets, the requirement for complicated drug-screening assays, and the presence of homologous domains recognizing short and degenerate sequence motifs.9Gerstberger S. Hafner M. Tuschl T. A census of human RNA-binding proteins.Nat. Rev. Genet. 2014; 15: 829-845Crossref PubMed Scopus (984) Google Scholar In their recent study, Ghidini et al.1Ghidini A. Cléry A. Halloy F. Allain F.H.T. Hall J. RNA-PROTACs: degraders of RNA-binding proteins.Angew. Chem. Int. Ed. Engl. 2021; 60: 3163-3169Crossref PubMed Scopus (29) Google Scholar reported a new type of PROTAC, termed RNA-PROTAC, that aims to degrade RBPs. Based on the established pre-let-7/Lin28 complex,11Loughlin F.E. Gebert L.F. Towbin H. Brunschweiger A. Hall J. Allain F.H. Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28.Nat. Struct. Mol. Biol. 2011; 19: 84-89Crossref PubMed Scopus (94) Google Scholar the authors utilized the key partial sequence (AGGAGAU) of pre-let-7 that binds to Lin28 as the POI ligand, together with a peptide ligand for E3 ligase von Hippel-Lindau (VHL) and a peptide linker to form the RNA-PROTAC ORN3P1 (Figure 1B). Because nucleotides can easily be degraded by nucleases, the authors replaced the phosphodiester backbone of nucleotide AGGAGAU with phosphorothioate linkages and alkylated the ribose 2′-hydroxyl group to enhance nuclease resistance and bioavailability. Because of the excellent water solubility of nucleotides, the peptide that contains the E3 ligand (LA[Hyp]YI: an optimized peptide with sufficient VHL-binding activity at a smaller size) and a peptidic linker (C[Ahx]) was chosen to coordinate the nucleotide's hydrophilicity, thereby facilitating chemical synthesis and purification. The nucleotide and peptide components were linked by an N-hydroxy succinimide (NHS)-based (NHS is a biochemical tool for bioconjugation) linker to generate ORN3P1. ORN3P1 selectively drove Lin28 degradation via a ubiquitination-proteasome mechanism and could inhibit the growth of tumor cell lines with negligible toxicity.1Ghidini A. Cléry A. Halloy F. Allain F.H.T. Hall J. RNA-PROTACs: degraders of RNA-binding proteins.Angew. Chem. Int. Ed. Engl. 2021; 60: 3163-3169Crossref PubMed Scopus (29) Google Scholar The RNA-PROTAC described in this recent study expands the PROTAC concept by introducing a short oligoribonucleotide as the POI ligand (Figure 1B). Taking advantage of endogenous regulatory networks between biomacromolecules should allow us to effectively degrade other disease-linked proteins. For instance, pathogenic DNA-binding proteins, such as dysregulated/mutated transcriptional factors and histones, control disease initiation and progression via regulating gene expression and are also difficult to target by traditional therapeutic methods.12Bushweller J.H. Targeting transcription factors in cancer - from undruggable to reality.Nat. Rev. Cancer. 2019; 19: 611-624Crossref PubMed Scopus (220) Google Scholar Thus, DNA-protein interactions might be targeted by the development of DNA-PROTACs. The establishment of RNA-PROTACs provides a new therapeutic approach to tackle the increasing evidence for the clinical relevance of RNA-protein machineries in human disease. Clinical application, however, will require further optimization of RNA-PROTAC structures. RNA-PROTACs have large molecular weights, which may influence their bioavailability. Therefore, the idea of in-cell click-formed proteolysis targeting chimeras (CLIPTACs), which comprise two distinct precursors that form an integrated PROTAC through "click chemical reaction" in cells, could be exploited to design a pair of smaller RNA-PROTAC precursors to improve cellular uptake.13Lebraud H. Wright D.J. Johnson C.N. Heightman T.D. Protein degradation by in-cell self-assembly of proteolysis targeting chimeras.ACS Cent. Sci. 2016; 2: 927-934Crossref PubMed Scopus (173) Google Scholar Additionally, the existing RNA-PROTACs use peptide fragments as E3 ligands and linkers, which are easy to digest and are inefficient at penetrating cell membranes. Therefore, water-soluble, non-peptidic linkers and E3 ligands might be considered to overcome these limitations and facilitate the synthesis of RNA-PROTACs. Finally, as RNA-PROTACs are inherently unstable in the presence of nucleases and/or proteases, nanoscale drug delivery systems might be introduced to protect RNA-PROTACs from decay, thereby increasing their biological efficiency in vivo.14Roberts T.C. Langer R. Wood M.J.A. Advances in oligonucleotide drug delivery.Nat. Rev. Drug Discov. 2020; 19: 673-694Crossref PubMed Scopus (427) Google Scholar This work was supported by the Science and Technology Foundation of Sichuan Province, China ( 2019JDTD0013 ), the 1.3.5 Project for Disciplines of Excellence , West China Hospital , Sichuan University ( ZYJC18030 and ZYGD20008 ), the National Natural Science Foundation of China ( 82073319 ), and the China Postdoctoral Science Foundation ( 2019T120849 ). The authors declare no competing interests.