Estrone Conjugated Oligonucleotides are short oligonucleotides covalently linked to the steroid hormone estrone. Oligonucleotides conjugated to estrone (E1) allow the design of therapeutic oligonucleotide-conjugates. Estrone is a ligand for estrogen receptors (alpha and beta) overexpressed in hormone-dependent cancers, including breast, ovarian, and endometrial cancers. Estrogen receptors are members of the nuclear receptor superfamily, and all members of this superfamily have a similar architecture.
When the estrone oligonucleotide conjugate binds to one of these receptors, it is internalized into the cell. As a steroid, estrone is hydrophobic or lipophilic, and attaching it to a hydrophilic oligonucleotide enables its interaction with lipid bilayers and penetration of the bilayer.
| Estrone-E1 3-Hydroxyestra-1,3,5(10)-trien-17-one (E1) | Estradiol-E2 17-β-estradiol (E2) | Testosterone 17ß-Hydroxyandrost-4-en-3-one |
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Figure 1: Chemical structures of estrone-E1, estradiol-E2, and testosterone.
Specifically targeting cells or tissues other than the liver for delivering therapeutic drugs has attracted growing interest in recent years. The delivery of negatively charged DNA or RNA oligonucleotides across the cell membrane and into the target tissue is the primary challenge in oligonucleotide therapeutics. However, the high polarity of polyanionic oligonucleotides and their relatively rapid nuclease-mediated cleavage are major pharmacokinetic hurdles to their in vivo application. The polyanionic architecture of oligonucleotides prevents the penetration of target cells or tissues.
Conjugating oligonucleotides to estrone enhances receptor-mediated uptake via increased lipophilicity. The conjugation of drugs to tissue-specific targeting molecules, such as estrone or cell-penetrating peptides (CPPs), enhances the delivery of therapeutic oligonucleotides, including small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), to specific tissues.
The synthesis of estrone-oligonucleotide conjugates typically requires modifying the estrone molecule to create a reactive handle, often by adding an amino group, that allows the attachment to the oligonucleotide.
Meschaninova et al. (2019) reported a solid-phase-based synthesis strategy utilizing N,N'-disuccinimidyl carbonate (DSC) activation for attaching estrone to the 5'-end of an oligonucleotide while still attached to the solid support (CPG). In this method, a DNA or RNA oligonucleotide chain is synthesized using standard automated phosphoramidite chemistry. Still, instead of adding a final nucleotide, the 5'-hydroxyl group of the oligonucleotide is activated using N,N'-disuccinimidyl carbonate (DSC). The amino-functionalized estrone derivative, estrone linked to an alkyl amine, is added to the column. The amino group reacts with the activated carbonate to form a stable carbamate linkage. The final conjugate is cleaved from the solid support and deprotected using standard ammonia or methylamine treatments.
Figure 2: Solid phase 5’-activation using DSC.
Figure 3: Solid phase 5’-functionalization of an oligonucleotide on the solid support.
Bang et al. (2013) synthesized two estrone-derived phosphoramidites, one with an octyl spacer. The researchers attached them to the 5′-end of the sense strand of a 21-mer small interfering RNA (siRNA) targeting vascular endothelial growth factors (VEGF) in an oligonucleotide synthesizer.
| Estrone phosphoramidite | Estrone-siRNA |
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Figure 4: Structures of estrone phosphoramidite and an estrone siRNA conjugate.
VEGF stimulates the formation of new blood vessels and increases vascular permeability. VEGF is overproduced in various diseases, including cancer. VEGF is a target for cancer therapies to starve tumors by inhibiting their blood supply.
Bang et al. synthesized the estrone phosphoramidite and directly incorporated it at the 5'-ends of the sense strand. The resulting estrone-conjugated siRNAs readily passed through cellular membranes and down-regulated the target protein with a specific distribution in vivo.
According to Rivas et al. (2005) and Friedrich & Aigner (2022), siRNA is incorporated into the RNAi-Induced Silencing Complex (RISC). After siRNA activation by removing the ‘sense’ or ‘passenger’ strand, the remaining ‘antisense’ or ‘guide’ strand directs RISC towards binding to the target mRNA, where AGO-2 in RISC mediates cleavage where the nuclease activity of AGO2 cleaves the complementary mRNA across from the 10th nucleotide of the guide siRNA strand, triggering further degradation of the mRNA by the cellular RNA degradation processes.
Hawner & Ducho (2020) reviewed the conjugation of modified oligonucleotides, including siRNA, to small molecules for cellular targeting. In particular, the uptake of oligonucleotides conjugated to small molecules enables them to cross the cell membrane via their receptor-binding affinity.
References
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Friedrich M, Aigner A. Therapeutic siRNA: State-of-the-Art and Future Perspectives. BioDrugs. 2022 Sep;36(5):549-571. [PMC]
Hara Y, Waters EM, McEwen BS, Morrison JH. Estrogen Effects on Cognitive and Synaptic Health Over the Lifecourse. Physiol Rev. 2015 Jul;95(3):785-807. [PMC]
Hawner M, Ducho C. Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules. Molecules. 2020 Dec 16;25(24):5963. [PMC]
Meschaninova MI, Novopashina DS, Semikolenova OA, Silnikov VN, Venyaminova AG. Novel Convenient Approach to the Solid-Phase Synthesis of Oligonucleotide Conjugates. Molecules. 2019 Nov 22;24(23):4266. [PMC]
Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F. Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids. 2014 Nov;90:13-29. [PMC]
Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ, et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol. 2005;12:340–349. doi: 10.1038/nsmb918. [PubMed]
VEGF
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