Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer

Abstract

As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non-oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre-miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre-miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.

Keywords: RNA; cancer; chemical biology; metastatic; nucleic acids.

Fig. 1.

Rational design of small molecules targeting microRNA-21 (miR-21). (A) Scheme of the processing of miR-21 hairpin precursors and its oncogenic function. Arrows indicate sites of nuclease cleavage. (B) The sequence-based design of monomeric (1) and dimeric (2) compounds targeting the pre–miR-21 3D structure via Inforna. (C) Compound 2 (Fig. 2) decreased miR-21 and increased pre–miR-21 levels in MDA-MB-231 cells. Data represent mean ± SEM (n ≥ 3). *P < 0.05; **P < 0.01 as determined by a two-tailed Student t test. (D) miRNA profiling of MDA-MB-231 cells treated with 1 or 2. Dotted lines represent a false discovery rate of 1% and variance of S₀(0.1).

Fig. 2.

Chemical structures of compounds used in this study. (Top, gray) Compounds that bind pre–miR-21 at the Dicer site to inhibit processing. (Middle, blue) Active (green) and inactive (orange) RNase L nuclease recruitment modules; an X-ray structure for compound 3 was solved and is shown in the ball and stick model (24). (Bottom, yellow) Active, inactive, and negative control compounds used in RNase L recruitment studies to enzymatically cleave pre–miR-21.

Fig. 3.

Selective cleavage of pre–miR-21 by a small-molecule recruiter of RNase L in MDA-MB-231 cells. (A) Compound 5 dimerizes RNase L onto pre–miR-21 to enzymatically cleave it. (B) Compounds 2, 5, and 7 decreased miR-21 levels in MDA-MB-231. RNase L recruiting 5 decreased pre–miR-21 while 2 and 7 increased pre–miR-21 levels. (C) Diminished effects of 5 (50 nM) on pre–miR-21 were observed upon siRNA ablation of RNase L. (D) Cotreatment of 2 with 5 increased pre–miR-21 levels. (E) Coimmunoprecipitation of RNase L showed a ∼3-fold increase in pre–miR-21 only with 5 (200 nM), while showing no enrichment of pre–miR-210. Data represent mean ± SEM (n ≥ 3). *P < 0.05; **P < 0.01 as determined by a two-tailed Student t test. (F) RT-qPCR profiling of miRNAs in MDA-MB-231 with 2 (1,000 nM) and 5 (50 nM) treatment exhibited high selectivity for miR-21. (G) The majority of down-regulated proteins with 5-treatment were proliferative proteins, while the majority of up-regulated proteins are anti-proliferative, as measured by global proteomics and pathway analysis. Dotted lines represent a false discovery rate of 1% and a group variance of S₀(0.1).

Fig. 4.

Compound 5 inhibits metastasis of TNBC in vivo. (A) In vivo treatment of 5 (10 mg/kg, q.o.d., 6 wk) decreased lung nodules (white nodules) stained with Bouin’s solution. (B) H&E staining of lung tissue from mice treated with 5 or vehicle. (C) Lung nodule tissue treated with 5, but not vehicle, exhibited decreased mature miR-21 and (D) pre–miR-21 expression, as measured by FISH probing. (E) Treatment with 5 increased PDCD4 levels in lung nodule tissue sections as determined by immunohistochemistry (IHC) staining. (Scale bar, 50 µm.) Data represent mean ± SEM (n ≥ 4). *P < 0.05; **P < 0.01 as determined by a two-tailed Student t test.