Precise Small Molecule Degradation of a Noncoding RNA Identifies Cellular Binding Sites and Modulates an Oncogenic Phenotype

Abstract

Herein, we describe the precise cellular destruction of an oncogenic noncoding RNA with a small molecule—bleomycin A5 conjugate, affording reversal of phenotype and a facile method to map the cellular binding sites of a small molecule. In particular, bleomycin A5 was coupled to a small molecule that selectively binds the microRNA-96 hairpin precursor (pri-miR-96). By coupling of bleomycin A5’s free amine to the RNA binder, its affinity for binding to pri-miR-96 is >100-fold stronger than to DNA and the compound selectively cleaves pri-miR-96 in triple negative breast cancer (TNBC) cells. Indeed, selective cleavage of pri-miR-96 enhanced expression of FOXO1 protein, a proapoptotic transcription factor that miR-96 silences, and triggered apoptosis in TNBC cells. No effects were observed in healthy breast epithelial cells. Thus, conjugation of a small molecule to bleomycin A5’s free amine may provide programmable control over its cellular targets. Few approaches are available to define the binding sites of small molecules within cellular RNAs. Our targeted cleavage method provides such an approach that is straightforward to implement. That is, we determined experimentally the site cleaved within pri-miR-96 in vitro and in cells; these studies revealed that the site of cleavage is the precise site for which the small molecule cleaver was designed and in agreement with modeling. These studies demonstrate the potential of sequence-based design to provide bioactive compounds that precisely recognize and cleave RNA in cells.

Small molecule recognition of disease-relevant RNA structures

“…The first example of using small molecule-bleomycin conjugates to cleave miRNAs came from Li et al, 177 in which they used a heterodimeric-bleomycin A5 conjugate to target oncogenic pri-miR-96. Both the bleomycin derivative and its site of conjugation to the small molecule were carefully selected.…”

“…47 To further improve bioactivity, a small molecule cleaver was synthesized by conjugating TGP-96 to bleomycin A5 (TGP-96-Bleo) via the C-terminal amine in the traditional DNA-binding domain of bleomycin A5, thus disrupting key interactions necessary for DNA recognition (Table S1, ESI †). 177 As bleomycin A5 has been shown to cleave AU base pairs, 175 and pri-miR-96 has AU base pairs in close proximity to TGP-96's binding site, this conjugation strategy had a high potential for success.…”

“…16B and Table S1, ESI †). 177 A control compound lacking the RNA-binding modules cleaved plasmid DNA at levels 5-fold greater than those seen with TGP-96-Bleo, 177 indicating conjugation of bleomycin A5 to RNA binding modules reduces its affinity for DNA, lowering the potential for off-targets. This was further supported by visualizing g-H2AX foci formation in cells, a marker for DNA double stranded breaks.…”

The Non-Coding RNA Landscape of Plasma Cell Dyscrasias

“…Liu et al then reported that the alkaloid sophocarpine antagonized Dicer-mediated processing of miR-21 [75]. Later on, a bleomycin A5 conjugate, which selectively inhibits Drosha processing of pri-miR-96, was discovered and found to upregulate the miR-96 pro-apoptotic target FOXO1, inducing breast cancer cell death [76]. Interestingly, Costales et al identified a SM with overlapping affinity for the precursors of both miR-515 and miR-885, which share a common target motif; this compound was optimized to bind only miR-515 by Inforna, a computational approach that drives sequence-based design of SM targeting structured RNA [77], leading to the Targaprimir-515 molecule [78].…”

Unveiling the druggable RNA targets and small molecule therapeutics

“…In 2018, Li et al described a novel small molecule, bleomycin A5 conjugate, which selectively binds to the precursor of miR-96 106 ( Fig. 2A).…”