Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor
miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASO), including morpholinos and \xce\xb3PNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.
Introduction<!>Bifunctional small molecule-oligonucleotide hybrid inhibits miR-21 biogenesis<!>The bifunctional approach can be used to quickly generate an inhibitor for another miRNA.<!>The activity of the bifunctional molecule is sensitive to the affinity of the RNA binding unit to pre-miRNA.<!>Using \xce\xb3PNA ASO improved the activity of the bifunctional molecule.<!>Conclusion<!>Chemicals and instrumentation<!>2-hydroxy-1,3-dioxo-N-(prop-2-yn-1-yl)isoindoline-5-carboxamide (11S)<!>N-(2-(2-(2-(hex-5-ynamido)ethoxy)ethoxy)ethyl)-2-hydroxy-1,3-dioxoisoindoline-5-carboxamide (11L)<!>Synthesis of PNA oligomers<!>Synthesis of bifunctional small molecule-ASO hybrid<!>Electrophoretic mobility shift assay<!>In vitro Dicer inhibition assay
<p>MicroRNAs (miRNAs) are short non-coding RNAs. They play a major regulatory role in orchestrating various biological processes1. Dysregulation of miRNAs is associated with many diseases. They have been considered as a new type of therapeutic target2. Therefore, a lot of efforts have been dedicated in developing approaches for regulating the function or biogenesis of miRNAs3–5. These methods are expected to find application in disease therapy and the study of miRNA function.</p><p>One promising direction for miRNA regulation is focused on developing small molecule inhibitors to regulate the biogenesis of miRNA. The pathway starts with the transcription of miRNA gene followed by RNase Drosha-mediated processing of the primary transcript (pri-miRNA) to produce hairpin looped structure, precursor miRNA (pre-miRNA). pre-miRNA was then processed by RNase Dicer to give the mature miRNA. Small molecules targeting these processes could potentially serve as miRNA inhibitors. For this purpose, intense researches have been focusing on identification of small molecules targeting miRNA precursors in order to disrupt miRNA maturation.3–4, 6–9 Despite the significant progresses in this field, it remains challenging to design or identify small molecules that selectively recognize the target RNAs while at the same time provide satisfactory biological activities.10–11</p><p>To address the issues that an RNA binder lacks desired activity, we recently reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer.12–14 These bifunctional molecules separate the functions of RNA recognition and RNA inhibition into two distinct structural units (i.e., a pre-miRNA binding unit and a Dicer inhibiting unit). The recognition of a target pre-miRNA by the RNA binding unit brings the conjugated weak Dicer inhibitor unit into the proximity to the pre-miRNA cleavage site of the bound Dicer molecule, thus blocking the enzymatic activity of Dicer and inhibiting the biogenesis of the target miRNA. We demonstrated that this bifunctional strategy significantly enhanced the inhibitory activity of the identified pre-miRNA binding small molecule (i.e., neomycin) against the maturation of miR-21, a well-known oncogenic miRNA overexpressed in many cancers.12, 14 However, aminoglycosides including neomycin are known to have poor selectivity in RNA recognition.10, 15 As a result, the neomycin-based pre-miRNA binding unit in the reported bifunctional molecules would not likely to provide desired selectivity.</p><p>Chemically modified antisense oligonucleotides (ASOs) have been developed to recognize and block the function of RNAs, including miRNAs, and have made significant contributions on RNA functional studies and therapeutic development for RNA-related diseases.16–24 Significant benefits of using ASOs for RNA targeting include enhanced recognition specificity and their sequence-based design that allows rapid generation of RNA binders/inhibitors when compared to the small molecule-based approaches. To provide a pre-miRNA binding unit with enhanced RNA recognition specificity in our bi-functional approach, in this work, we investigated the possibility of using the ASO to replace a small molecule as the RNA targeting unit (Fig. 1). We designed bifunctional molecules with different ASOs conjugated to weak Dicer inhibitors to achieve the inhibition of miR-21 maturation through Dicer inhibition.</p><!><p>Morpholino ASOs bind to complementary RNAs in high affinity, are resistant to nucleases and have been widely used for knocking down gene expression, modifying RNA splicing and inhibiting miRNA function and maturation.25–28 These favorable characteristics make morpholino ASO a promising candidate to be used as the pre-miRNA targeting unit in our bifunctional design. Pre-miRNAs typically fold to form stem-loop structures, which provide exposed single-stranded loop regions to be targeted by ASOs. The lack of negative charges on morpholino allows it to invade into the stem region once morpholino binds to the exposed loop sequences.29–30 Morpholino ASOs of 25-mer or longer are commonly used to ensure satisfactory inhibition. However, a longer length ASO can potentially result in increased off-target binding due to the imperfect pairing between partial morpholino sequences and different cellular RNA sequences under physiological conditions.29 Instead of requiring ASOs to satisfy both recognition and inhibitory functions, we expect the bifunctional design would alleviate the inhibitory contribution from ASOs (which can be compensated from the Dicer inhibiting unit), therefore, allowing us to use a shorter length, which may be beneficial in reducing off-targeting inhibition and improving cellular delivery. With the high melting points of a typical 25- and 14-mer morpholino ASOs around 90 oC and 60 oC,31 we expect a short morpholino ASO should provide sufficient binding affinity to RNAs.</p><p>With the above considerations in mind, we designed a set of morpholino ASOs with varying lengths targeting the loop region of pre-miR-21 (Fig. 2A). To check the binding activity of morpholino ASOs to pre-miR-21, we performed electrophoretic mobility shift assay (EMSA). 32P-labeled pre-miR-21 was prepared by in vitro transcription. Its electrophoretic mobility in the presence of various ASOs was then determined by using non-denaturing polyacrylamide gel and phosphor imaging. A reduced mobility rate of pre-miR-21 indicated a complex formed through the binding between the ASO and pre-miR-21. As shown in Fig. 2B, morpholino ASO 6, a positive control 25-mer morpholino molecule designed to target part of the loop region and the full mature miR-21 sequence in the stem region of pre-miR-21 (commercially available from Gene Tools, LLC), reduced the mobility of pre-miR-21 significantly, while morpholino ASO 7, a negative control designed to target a different RNA sequence (pre-miR-29b-1) with the same length as that of 6, did not show any shifting indicating no binding to pre-miRNA-21. These results showed that the recognition by morpholino ASOs was sequence-specific and the assay was working as expected. Importantly, morpholino ASOs of 14-mer and longer (3-5) were also found to reduce the mobility of pre-miR-21, indicating their strong binding to pre-miR-21. It's worth noting that 5 and 6 exhibited incomplete band shifting compared to the shorter ASOs 3 and 4, suggesting the increased difficulty in invading the stem region. These results led us to use 3, the shortest morpholino ASO that still showed strong binding to pre-miR-21 as the pre-miR-21 binding unit in the bifunctional molecules in the following study.</p><p>As a member of the endoribonuclease III family, Dicer processes double-stranded RNA in a mechanism similar as other RNases in the family do.32–33 2-hydroxyisoindoline-1,3-dione (Fig. 2C, 8) was reported as a weak inhibitor for other endoribonuclease III family enzymes.34 We anticipated that it could serve as a Dicer inhibiting unit in the bifunctional small molecule-ASO hybrids. To produce Dicer inhibition units that could be conjugated to the pre-miRNA binding ASOs through click chemistry, we modified 8 to give two alkyne-appended inhibitors with short and long linkers (Fig. 2D, 11 S & L). The synthesis starts with the acylation of 10 S & L by 9 (Fig. 2D). The intermediates were then reacted with hydroxyl amine to give the alkyne modified inhibitors (11 S & L). The bifunctional small molecule-ASO hybrids were finally prepared by click reaction between 11 and the azide-modified ASO molecules (Fig. S1, commercially available from Gene Tools, LLC).</p><p>To evaluate the activity of the bifunctional molecules in inhibiting the maturation of pre-miR-21, we performed an in vitro pre-miR-21 processing assay as previously reported.12 32P-labeled pre-miR-21 was incubated with recombinant human Dicer enzyme in the absence or presence of different compounds and the cleavage of pre-miR-21 was then monitored by using denaturing polyacrylamide gel electrophoresis and phosphor imaging. As show in Fig. 2E & F, Dicer can efficiently cleave pre-miR-21 to form mature miR-21 (Lane 2). In the presence of positive control 25-mer morpholino ASO 6 (10 μM), the cleavage of pre-miR-21 was significantly inhibited (Lane 3). Encouragingly, the bifunctional molecule 3A inhibited the cleavage of pre-miR-21 almost completely at 10 μM concentration (Lane 8), while the two functional units alone (Lane 4 for ASO 3 and Lane 5 for 11S), only showed background level of inhibition at the same condition. A comparison of the activity of 3A and 3B showed that the inhibition potency of the bifunctional molecule decreased as the linker length between the two functional units increased. This observation is also mirrored in our previous study on bifunctional small molecule miRNA inhibitors12. We also confirmed the binding of 3A to pre-miR-21 by EMSA (Fig. 2B Lane 8). Taken together, these results demonstrated that conjugation of pre-miR-21 targeting ASO molecule to the Dicer inhibitory unit significantly enhances the Dicer inhibition potency and that a proper linker length between the two units is critical for the activity.</p><!><p>With the modular design of the bifunctional strategy, we investigate if a new inhibitor for another miRNA can be quickly generated simply by switching the pre-miRNA binding ASO in the bifunctional small molecule-ASO hybrid molecule. To test this, miR-29b-1, whose dysregulation is associated with dysfunctional immunity in cancer,35 was chosen as a target. We designed a 14-mer morpholino ASO 12 (Fig. 3A) as pre-miR-29b-1 targeting unit targeting the loop region matching that of ASO 3. The azide-modified ASO 12 was conjugated to 11S through click chemistry to give bifunctional molecule 12A (Fig. 3B). The 25-mer ASO 7 designed to inhibit miR-29b-1 maturation was used as the positive control. The Dicer-mediated pre-miR-29b-1 cleavage assay was then performed to evaluate the inhibitory activity of these compounds. As shown in Fig. 3C & D, pre-miR-29b-1 was completely cleaved into mature miR-29b-1 by Dicer. In the presence of ASO 7 (50 μM), the cleavage reaction was partially inhibited. No inhibition was observed when using individual units ASO 12 (50 μM) and 11S (1 mM). In contrast, the bifunctional conjugate 12A (50 μM) was able to inhibit the reaction almost completely under the same concentration as ASOs 7 and 12. The results demonstrated that the bifunctional approach could be readily used to generate different miRNA inhibitor by simply changing the RNA targeting unit.</p><!><p>To improve the activity of bifunctional inhibitors, we studied the contribution from each functional unit in the bifunctional molecule. We first investigated the effect of the pre-miRNA binding unit on the activity of the bifunctional molecule. We synthesized another two bifunctional molecules 1A and 2A (Fig. 2A & D), containing the same Dicer inhibiting unit as in 3A but using shorter (10- and 12-mer) morpholino ASOs as the pre-miR-21 binding unit. We then tested the activity of 1A to 3A toward the inhibition of pre-miR-21 cleavage using the in vitro pre-miR-21 processing assay. As shown in Fig. 4, the inhibition of pre-miR-21 cleavage decreased dramatically when the length of morpholino ASO decreased. This observation clearly demonstrated that the activity of the bifunctional molecules is critically dependent on the affinity of the RNA binding unit to the pre-miRNA. Inspired by this discovery, we asked whether more potent bifunctional inhibitors could be obtained by replacing morpholino ASO with even more potent class of as our RNA targeting unit.</p><!><p>Peptide nucleic acids (PNAs) are synthetic DNA/RNA mimics that have been reported to have higher affinity in DNA/RNA binding when compared to morpholino.36 γPNAs contain a charge-neutral N-(2-aminoethyl) glycine backbone (Fig. S2) and are conformationally pre-organized into a right-handed helical motif due to the (R)-stereogenic center at the backbone.37 The unique structural characteristics allow them to have superior properties including increased binding affinity to nucleic acid, high solubility, and improved biocompatibility.37–38 To test whether γPNA could be used as the RNA targeting unit in our bifunctional design, we synthesized three γPNAs with different lengths (7- to 11-mer) targeting the loop region of pre-miR-21 (ASO 13-15, Fig. 5A). A 12-mer ASO 16 with a random sequence was also prepared and used as a negative control. We first examined their binding to pre-miR-21 by EMSA. As shown in Fig. 5B, ASO 16 failed to cause band shift as expected. In contrast,9- and 11-mer γPNA ASOs 14 and 15 reduced the mobility of the pre-miR-21 band significantly indicating their strong binding to pre-miR-21. The short 9-mer 13 was not able to cause the shift of pre-miR-21 band, likely due to the decreased binding affinity to pre-miR-21. Further studies showed that 14 and 15 were able to shift pre-miR-21 band at a concentration as low as 500 nM (Fig. S3), which is much lower than that required for morpholino ASO 3, consistent with their stronger binding affinity to RNAs. These results led us to choose 15 as the RNA targeting unit to form the γPNA-based bifunctional small molecule-ASO hybrid. We previously reported that compound 17 (Fig 5C) inhibits the activity of Dicer with an IC50 of around 100 μM,12 which is a more potent Dicer inhibitor in comparison to 11S that had no inhibition at 1 mM concentration (Fig 2E Lane 5). With an expectation that using a more potent Dicer inhibiting unit may further improve the activity of the bifunctional small molecule-oligonucleotide hybrid, we conjugated compound 17 with azide-modified 15 and obtained bifunctional small molecule-γPNA ASO hybrid 15A (Fig. 5C & S2). As another comparison, conjugate 3C was also prepared (Fig. 5C), which has the same Dicer inhibiting unit as in 15A but using 14-mer morpholino 3 as the RNA binding unit. The inhibitory activities of these compounds were then studied using the in vitro pre-miR-21 processing assay and the IC50 value for each compound was obtained. As shown in Fig. 5D, 15A inhibited the cleavage reaction in a dose-dependent manner with an IC50 of 0.5 μM. Compared to the morpholino counterpart 3C (IC50 3.3 μM), the γPNA moiety in 15A improved the activity of the conjugate significantly. These results demonstrated that γPNA may be a superior RNA binding unit that greatly enhance the inhibitory activity of the bifunctional small molecule-ASO hybrid. Surprisingly, comparing the two morpholino-containing bifunctional hybrids 3C (with a more potent Dicer inhibitor 17) and 3A (IC50 4.3 μM, bearing a weaker Dicer inhibitor 8), 3C only exhibited slight improvement on the activity in inhibiting pre-miR-21 cleavage (Fig. 5D). We suspect that due to the unique strategy of the bifunctional approach, which inhibits Dicer-mediated pre-miRNA cleavage by increasing the local effective concentration of a weak Dicer inhibitor at the pre-miRNA cleavage site through the targeting of the RNA binding unit, the contribution from the potency of the inhibition unit may not be as significant because of the local enrichment of the inhibition unit. However, the detailed reason remains to be investigated.</p><!><p>We previously demonstrated that using bifunctional small molecules to achieve enzymatic inactivation of Dicer can be a novel way for miRNA inhibition. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to and locally enriched at the cleavage site of specific pre-miRNA to block Dicer-mediated pre-miRNA processing. To expand on this bifunctional strategy, we showed that ASO molecules, including morpholinos and γPNAs, could be readily used as the RNA targeting unit to replace their small molecule counterparts and generate inhibitors against miRNAs. Our studies also showed that the potency of the bifunctional small molecule-ASO hybrids is mainly determined by the binding of the RNA targeting ASO molecules. Importantly, the ASO molecules used in this approach were significantly shorter than the commonly used anti-miRNA ASOs, which may provide benefits in specificity and cellular delivery.20, 29, 39–40 The current form of bifunctional hybrids were unfortunately not cell permeable (data not shown) and require further optimization. Nevertheless, we believe that these studies should contribute to the development of new classes of miRNA inhibitors.</p><!><p>Morpholino ASOs were purchased from GeneTools, LLC. Other reagents were purchased from Sigma-Aldrich or Alfa Aesar. Column chromatography was carried out on silica gel (pore size 60 Å, 200–425 mesh particle size, Sigma Aldrich). HPLC was performed using a Thermo Scientific UltiMate 3000 semi-preparative system. 1H and 13C NMR spectra were recorded on a Bruker Avance III 300 spectrometer. Chemical shifts are reported in parts per million (ppm, δ) referenced to the residual 1H resonance of the solvent.41 Mass spectra were obtained with a Waters Micromass mass spectrometer with electrospray ionization probe.</p><!><p>Compound 9 (192 mg, 1 mmol) was dissolved in SOCl2 (2 mL). The mixture was refluxed for 2 h and vacuumed to remove SOCl2. The residue was then dissolved in THF (5 mL) and the reaction was cooled to 0 °C. Propargylamine (10S, 55 mg, 1 mmol) was added dropwise followed by Et3N (121 mg, 1.2 mmol) at 0 °C. The reaction was stirred for 3 hours at room temperature. The solvents were then removed by vauum. To the residue was added NH2OH·HCl (70 mg, 1 mmol) and pyridine (10 mL). The mixture was stirred at 90 °C for overnight. After removing the solvents by vacuum, the residue was partitioned between water (20 mL) and ethyl acetate (20 mL). The organic phase was dried with Na2SO4 and concentrated by vacuum. The crude product was then purified by chromatography (hexane/ethyl acetate = 1:1) to give an off-white solid (182 mg, yield 75%). 1H NMR (DMSO-d6, 300 MHz): 10.93 (s, 1H), 9.34–9.30 (t, J= 10.8 Hz, 1H), 8.29–8.25 (m, 2H), 7.95–7.92 (d, J= 7.5 Hz, 1H), 4.11–4.08 (q, J= 7.8 Hz, 2H), 3.17–3.16 (t, J= 5.1 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz): 164.15, 163.53, 163.46, 138.97, 133.65, 131.068, 129.11, 123.16, 121.31, 80.73, 73.21, 28.78. HRMS (m/z) found (calc.) for C12H8N2O4 (M): [M+H]+, 245.0560 (245.0562).</p><!><p>11L was made as the preparation of 11S except that compound 10L42 was used to replace 10S (Yield: 35 %). 1H NMR (MeOD, 300 MHz): 8.27–8.25 (m, 2H), 7.95–7.92 (d, J= 7.8 Hz, 1H), 3.71–3.63 (m, 8H), 3.58–3.54 (t, J= 10.5 Hz, 2H), 3.37–3.33 (t, J= 12.9 Hz, 2H), 2.33–2.28 (t, J= 14.7 Hz, 2H), 2.24–2.18 (m, 3H), 1.83–1.74 (m, 2H). HRMS (m/z) found (calc.) for C21H25N3O7 (M): [M+Na]+, 454.1588 (454.1590). Compound 17 were prepared as reported previously.12</p><!><p>Fmoc-protected γPNA monomers were synthesized as previously reported.43 All oligomers were synthesized on solid-support using standard Fmoc-chemistry procedures. The oligomers were cleaved from resin using an m-cresol: trifluoroacetic acid (TFA) (5:95) cocktail solution and precipitated with ether. PNA oligomers were purified by reverse-phase high performance liquid chromatography in analytical mode (C18 column with dimensions 4.6 mm X 250 mm, 1 mL/min, 60 oC) or in semi-preparative mode (C18 column with dimensions 19 mm X 100 mm, 5 mL/min, 55 oC), and characterized by MALDI-TOF MS with CHCA matrix. All PNA stock solutions were prepared using nanopure water, and the concentrations were determined at 90 oC on a Agilent Cary UV-Vis 300 spectrometer equipped using the following extinction coefficients: 13,700 M−1cm−1 (A), 6,600 M−1cm−1 (C), 11,700 M−1cm−1 (G) and 8,600 M−1cm−1 (T).</p><!><p>To a mixture of ASO-N3 (50 nmol in 50 μL of H2O) and 11S/L (100 nmol in 50 μL of DMSO) was added Copper (I) iodide (50 nmol in 10 μL of H2O), sodium ascorbate (100 nmol in 10 μL of H2O) and Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (50 nmol in 10 μL of DMSO). The reaction was stirred at room temperature for 2 h. The product was purified by HPLC and characterized by MS (Table S1).</p><!><p>32P-labeled pre-miRNA were prepared as described before.13 Individual tested compound (200 μM or as specified) was incubated with 32P-labeled pre-miR-21 (around 100 nmol) in buffer (HEPES 24 mM, NaCl 200 mM, EDTA 0.04 mM, MgCl2 2.5 mM, ATP 1 mM, pH 7.5) at 37 °C for 1.5 h. The mixture was then subjected to 15% non-denaturing polyacrylamide gel electrophoresis at 4 °C. The gel was visualized by phosphor imaging and analyzed by Quantity One software (Bio-rad).</p><!><p>32P-labelled pre-miRNA was prepared as described above. A 10 μL of the reaction mixture was made by incubating 32P-labeled pre-miRNA (1 μL, ~20 ng) with 1 μL of Dicer enzyme (Genlantis) and various compounds in buffer (HEPES 24 mM, NaCl 200 mM, EDTA 0.04 mM, MgCl2 2.5 mM, ATP 1 mM, pH 7.5) at 37 °C for 2.5 h. The reaction was stopped by boiling with an equal volume of Gel Loading Buffer II (Thermo-Fisher Scientific) for 5 min. The non-cleaved pre-miRNA and the mature miRNA were resolved by 18% denaturing polyacrylamide gel. The gel was analyzed by phosphorimaging and quantified by Quantity One software (Bio-rad). The IC50 was obtained by fitting the data with Origin software.</p>