A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21 Precursor and Suppresses Dicer Processing

Abstract

MicroRNAs (miRNAs) help orchestrate cellular growth and survival through post-transcriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects, chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers leading to the down regulation of key tumor suppressing and pro-apoptotic factors; suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic β-hairpin peptidomimetics which binds to RNA stem loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with a KD=200nM and prefers it over other pre-miRNAs. The NMR structure of the complex shows the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rational for the peptide binding activity and clear guidance for further improvements in affinity and targeting.

Visualizing a protonated RNA state that modulates microRNA-21 maturation

“…1c). This observation is consistent with previous NMR studies on miR-21 precursor 33,34 , where mutations of the pre-element were made to quench the structural flexibility into a single conformational state 33 . When we carried out an NMR 13 C-1 H HSQC experiment that probes nonsolvent-exchangeable signals, surprisingly only 20 out of a total of 29 expected NMR resonances from preE-miR-21 were observed at room temperature ( Fig.…”

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

“…Comparison of the top 20 NMR solutions confirms these as stable features of the molecule. NMR study of pre-miR-21 revealed weak signals corresponding to two tandem U-G/G-U pairs at the apical junction whereas the 14-nt apical loop is otherwise unstructured 31 .…”

“…1a). RNA secondary structure prediction programs tend to include base pairs in relatively long loops that are not necessarily stable 31,32 . We disregarded 1 or 2 base pairs that are separated from the hairpin stem on both strands.…”

“…Our understanding of miRNA hairpins is mostly based on secondary structure predictions 29 , sometimes assisted by chemical or nuclease footprinting 30 . We are interested to determine threedimensional structures of apical junctions and loops for their important roles in miRNA maturation and regulation 5,7,8,10 and as targets for drug discovery 31 . Apical junctions and loops are shared between pri-miRNA and pre-miRNA, but here for simplicity we use pri-miRNA to name individual apical junctions and loops.…”

“…Apical junctions and loops are shared between pri-miRNA and pre-miRNA, but here for simplicity we use pri-miRNA to name individual apical junctions and loops. To date only two apical stem-loops have been structurally characterized, using NMR spectroscopy, with pri-miR-20b folding to well-defined rigid structures 32 and pri-miR-21 unstructured 31,33 . The human genome encodes 1,881 pri-miRNA hairpins that differ from each other greatly in terms of both their sequences and predicted secondary structures 29 .…”