Defining RNA–Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs

CD4⁺ T cell responses and the induction of IL-12 family genes after infection with <i>L. monotytogenes</i>.

<p><i>A</i>, C57BL/6 mice were intravenously infected with 2.5×10⁴ Lm-Ova on day 0, and splenocytes were obtained on day 7. CD4⁺ T cells expressing IFNγ and IL-17 were measured by intracellular staining after stimulation with LLO_190–201. <i>B and C</i>, Bone marrow-derived DC or macrophages were stimulated with LPS, Pam₃CSK₄, or irradiated Lm-Ova for four hours. Cells were harvested and analyzed for the mRNA expression of IL-12 family genes by using RT-PCR (<i>B</i>, bone marrow-derived DC), or quantitative real-time PCR analysis (<i>C</i>). Values are mean ± SD. Data shown represent two independent experiments.</p

Increased pathogen-specific Th17 responses in the absence of IL-12p35 and IL-27EBI3.

<p>C57BL/6 (WT) or the indicated strains of mice (n = 5 per group) were intravenously infected with 2.5×10⁴ Lm-Ova on day 0. Seven days later, lymphoid cells from the spleen were obtained and CD4⁺ T cells expressing IFNγ and IL-17 were measured by intracellular staining after stimulation with LLO_190–201<i>(A and B)</i>. The lymphoid cells from the spleen were stimulated with LLO_190–201 peptide for three days, and the concentrations of IFNγ, IL-17 and IL-22 in the supernatant were measured by ELISA <i>(C)</i>. Bars in B are mean ± SEM. Values in <i>C</i> are mean ± SEM. Data shown are representative of two independent experiments. *,p<0.05; **,p<0.01 in comparison with WT group. #,p<0.05; ##,p<0.01 in comparison with p35^−/− group.</p

IL-17 and IL-22 cooperatively promote protective immunity against <i>L. monocytogenes</i> infection in p35^−/− mice.

<p><i>A</i>, C57BL/6 (WT) or groups of p35^−/− mice (n = 5 per group) were intravenously infected with 2.5×10⁴ Lm-Ova on day 0. Some of the p35^−/− mice were i.p. injected with 1 µg of recombinant IL-17, IL-22, or both on day 0, 2, 4. Seven days after the infection, bacterial burden in the livers of the infected mice was determined by measuring colony-forming unit. Bars are mean values. *, p<0.05 in comparison between two indicated groups.</p

p35^−/−EBI3^−/− mice are resistant to the infection with <i>L. monocytogenes</i>.

<p>C57BL/6 (WT) or the indicated strains of mice (n = 4–5 per group) were intravenously infected with 2.5×10⁴ Lm-Ova on day 0. Three (<i>A</i>) or seven (<i>B</i>) days later, the bacterial burden in the livers of the infected mice was analyzed by measuring colony-forming unit. Bars are mean values. Data shown are representative of three independent experiments. *, p<0.05 and **, p<0.01 in comparison between two indicated groups.</p

Enhanced production of Th17 cytokines in the p35^−/−EBI3^−/− mice during the early phase of infection with <i>L. monocytogenes</i>.

<p>C57BL/6 (WT) or the indicated strains of mice (n = 3 per group) were intravenously infected with 2.5×10⁴ Lm-Ova on day 0. Two to three days later, lymphoid cells were analyzed for the production of IFNγ by NK and NKT cells (<i>A</i>), or for the frequency of CD11b⁺Ly6C⁺ Tip DC (<i>B</i>). The production of IFNγ, IL-17, IL-17F and IL-22 by the splenocytes obtained three days after the infection was measured (<i>C</i>). Values are mean ± SD. Data shown are representative of two independent experiments. *,p<0.05; **,p<0.01 in comparison with WT group.</p

A role for IL-17 and Th17 cells on the resistance of p35^−/−EBI3^−/− mice against <i>L. monocytogenes</i> infection.

<p><i>A</i>, C57BL/6 (WT) or the indicated strains of mice (n = 4 per group) were intravenously infected with 2.5×10⁴ Lm-Ova on day 0. The mice were i.p. injected with 100 µg of anti-IL-17 or rat IgG on day 0, 2, 4. Seven days after the infection, bacterial burden in the livers of the infected mice was determined by measuring colony-forming unit. *, p<0.05 in comparison with rat IgG-treated group. <i>B and C</i>, IL-17F<i>^rfp</i> reporter mice were s.c. immunized with LLO peptide emulsified in CFA. Seven days later, lymphoid cells from spleen and draining lymph nodes of the immunized mice were isolated and stimulated with the LLO peptide in the presence of IL-23 (50 ng/ml), IL-1β (10 ng/ml) and anti-IFNγ for 5 days. CD4⁺ RFP⁺ cells were isolated by flow cytometry, and the expression of IL-17 and IFNγ was measured by intracellular staining (<i>B</i>). The sorted Th17 cells (5×10⁵ cells per transfer) were i.v. transferred into p35^−/− mice, followed by i.v. infection with 2.5×10⁴ Lm-Ova. WT or p35^−/− mice without the cell transfer were used as controls. Seven days after infection, bacterial burden in the liver was measured (<i>C</i>). Data shown are representative of two independent experiments. *, p<0.05 in comparison with p35^−/− mice without Th17 cell transfer.</p