Selective Small-Molecule Targeting of a Triple Helix Encoded by the Long Noncoding RNA, MALAT1

Abstract

Metastasis-associated lung adenocarcinoma transcript 1 ( Malat1/ MALAT1, mouse/human), a highly conserved long noncoding (lnc) RNA, has been linked with several physiological processes, including the alternative splicing, nuclear organization, and epigenetic modulation of gene expression. MALAT1 has also been implicated in metastasis and tumor proliferation in multiple cancer types. The 3' terminal stability element for nuclear expression (ENE) assumes a triple-helical configuration that promotes its nuclear accumulation and persistent function. Utilizing a novel small molecule microarray strategy, we identified multiple Malat1 ENE triplex-binding chemotypes, among which compounds 5 and 16 reduced Malat1 RNA levels and branching morphogenesis in a mammary tumor organoid model. Computational modeling and Förster resonance energy transfer experiments demonstrate distinct binding modes for each chemotype, conferring opposing structural changes to the triplex. Compound 5 modulates Malat1 downstream genes without affecting Neat1, a nuclear lncRNA encoded in the same chromosomal region as Malat1 with a structurally similar ENE triplex. Supporting this observation, the specificity of compound 5 for Malat1 over Neat1 and a virus-coded ENE was demonstrated by nuclear magnetic resonance spectroscopy. Small molecules specifically targeting the MALAT1 ENE triplex lay the foundation for new classes of anticancer therapeutics and molecular probes for the treatment and investigation of MALAT1-driven cancers.

Figure 1.

HTS identification of compounds 5 and 16 as MALAT1 ENE triplex-binding ligands. (a) Schematic of SMM screening. Libraries of small molecules are spatially arrayed and covalently linked to a functionalized glass surface. For screening, fluorescently labeled Malat1 triple helix was incubated with the slides. After incubation, slides were washed to remove unbound oligonucleotide and imaged at 635 nm. (b) Pipeline developed to identify and validate compounds 5 and 16. (c) RT-qPCR of relative Malat1 levels in MMTV-PyMT tumors organoids with treatments of mock, DMSO, and indicated compounds (final concentration of 1 μM) and Malat1 antisense oligo (As) (final concentration of 200 nM). n = 3 biological replicates; bars represent plus or minus the standard error of the mean (SEM). (d) The structures of compounds 5 and 16.

Figure 2.

Reduction of Malat1 levels and organoid branching by compounds 5 and 16. (a) Image of organoids from MMTV-PyMT tumors after 7 days of culturing. The data for compound 5 are shown. (b) Relative Malat1 levels in MMTV-PyMT tumor organoids with treatments of Mock, DMSO, compounds 5 and 16 (final concentration of 1 μM), and Malat1-ASO (final concentration of 200 nM). n = 3 biological replicates; bars represent plus or minus SEM. (c) Relative organoid branching rate of MMTV-PyMT tumor organoids with treatment of mock, DMSO, compounds 5 and 16 (final concentration of 1 μM), and Malat1-ASO (final concentration of 200 nM). n = >80 organoids from 3 biological replicates; bars represent plus or minus the SEM. Single asterisks indicate P < 0.05, double asterisks indicate P < 0.01, and triple asterisks indicate P < 0.001 by Student’s t test.

Figure 3.

Modulation of Malat1 downstream genes by compound 5. (a) Relative RNA level of Krt16 in MMTV-PyMT tumor organoids with treatment of mock, DMSO, 1 and 0.5 μM of 5, and Malat1-ASO (final concentration of 200 nM). n = 3 biological replicates; bars represent plus or minus SEM. Single asterisks indicate P < 0.05 and double asterisks indicate P < 0.01 by Student’s t test. (b) Relative RNA level of Csn2 in MMTV-PyMT tumor organoids with treatment of mock, DMSO, 1 and 0.5 μM of 5, and Malat1-ASO (final concentration of 200 nM). n = 3 biological replicates; bars represent plus or minus SEM. Triple asterisks indicate P < 0.001 by Student’s t test.

Figure 4.

Induction of opposing structural responses in the ENE triple helix by compounds 5 and 16. (a) EFRET signal of M1^ET in the presence of vehicle (DMSO) alone reveals the conformational landscape of this triplex across multiplexed ionic conditions. The folded triplex has a higher E_FRET than partially unfolded triplex. The triplex conformational landscape is raised in the presence of (b) 10 μM compound 5, and primarily lowered in the presence of (c) 50 μM compound 16. In panels a–c, E_FRET was evaluated as a function of total monovalent concentrations (equimolar KCl and NaCl) from 2.6–202.6 mM and magnesium concentrations from 0.1–1 mM. All experiments were performed in triplicate (3 independent experiments), but a single set of 8 × 8 experiments is represented in each panel. (d) Secondary structure of M1^ET (ENE in cyan and Tail in blue) with the Cy3 and Cy5 positions illustrated by red and blue circles, respectively. Difference plots of E_FRET in the presence and absence of (e) 10 μM compound 5 and (f) 50 μM 16. Difference plots were calculated by subtracting E_FRET values in panel a from the values in panels b and c, respectively. An average of difference plots performed in triplicate (three independent experiments) is plotted in panels e and f. Compound 5 addition increases E_FRET (green shading), while compound 16 decreases E_FRET (brown shading) under all salt conditions.

Figure 5.

Evaluating compounds 5 and 16 binding to the MALAT1 ENE by FRET and ITC. (a) Compound 5 titration monitored by changes in E_FRET in 0.1 mM MgCl₂ and 2.6 mM total monovalent salt. The fit yields K_d = 2.3 ± 1.7 μM. (b) Compound 16 titration monitored by changes in E_FRET in 0.1 mM [Mg²⁺] and 52.6 mM total monovalent salt. The fit yields K_d = 6.1 ± 1.8 μM. (c) ITC analysis using compound 5 (500 μM) and wild-type M1^TH (75 μM) in a 1 mM MgCl₂, 200 mM monovalent salt, and 1% DMSO in 20 mM HEPES-KOH; pH of 6.9 indicates K_d = 2.9 ± 1.6 μM. (d) ITC analysis using compound 16 (500 μM) and wild-type M1^TH (75 μM) in a buffer containing 1 mM MgCl₂, 50 mM monovalent salt, and 1% DMSO in 20 mM HEPES-KOH; pH of 6.9 indicates K_d = 6.1 ± 2.1 μM. Error bars in all panels represent standard deviation of three independent experiments for all experiments except in panel d, which represents the standard error of the mean for two experiments.

Figure 6.

Molecular docking of compounds 5 and 16 to the MALAT1 ENE triplex core crystal structure. Three-dimensional structures are shown in a transparent surface and gray ribbon representation (PDBID: 4PLX). (a) MALAT1 sequence, within which the ENE is boxed. (b) The distribution of 246 independent dockings, shown as green van der Waals spheres, for compound 5 reveals a preference for major groove binding. (c) Highest populated docking cluster (and lowest energy) for compound 5 shown in surface representation buried within the triplex. (d) The interactions with the backbone phosphates and bases of nucleotides U10, U11, and C12 of the triplex Hoogsteen strand. Longer-range interactions are made with backbone phosphates of U37, U38, and U39 on the triplex Watson–Crick strand. The C11, C13, C14, and C15 atoms of compound 5 are shown as green CPK spheres interacting explicitly with U10, U11, and C12 of the triplex Hoogsteen strand, consistent with saturation transfer difference (STD) NMR experiments (see Figure 8). (e) Distribution of 156 dockings of compound 16 indicates primarily surface or minor groove interactions. (f) Cluster distribution analysis for compound 16 shows the highest populated cluster (44% of dockings) is located in a superficial pocket formed by the G48−G49 bulge. (g) Within this “bulge cleft”, compound 16 is positioned to make backbone phosphate hydrogen bond interactions with A67 and carbonyl nucleobase hydrogen bond interactions with U47 (purple arrows). The solvent accessibilities of compounds 5 and 16 are shown as gray densities in the 2D interaction plots.

Figure 7.

No effect on Neat1 levels in organoids treated with compounds 5 and 16. (a, b) Secondary structures of MALAT1 ENE and lncRNA NEAT1 triple helices, respectively. The pair of regions highlighted are significantly different between, namely (i) the bulge nucleotide region (red circle) and (ii) the number of U·AU base triples (blue circle). It unclear from the secondary structure if a cleft (binding site for compound 16 on the MALAT1 ENE triplex) is present in the NEAT1 structure and a crystal structure of the NEAT1 triplex is unavailable. Additionally, the deletion of two U·AU base triples within the triple helix of NEAT1 render a much shorter potential U·AU region (binding site for compound 5 on MALAT1 ENE triplex). (c) RT-qPCR of relative Neat1 levels in MMTV-PyMT tumors organoids with treatments of mock, DMSO, and compounds 5 and 16 (final concentration of 1 μM). n = 3 biological replicates; bars represent plus or minus SEM. Ns refers to no statistically significant differences in RNA levels.

Figure 8.

Saturation transfer difference (STD) NMR confirmation of compound 5 specificity for the MALAT1 ENE triple helix. (a) 1D ¹H NMR spectrum for compound 5 alone. (b–d) STD spectra for compound 5 following incubation with the ENE triplexes of MALAT1, NEAT1, and KSHV PAN, respectively. The protons on the para-methoxy aryl ring (highlighted in red) appear as two doublets at 7.12 and 7.42 ppm, while the proton on methyl substituted imidazole (highlighted in blue) appears as a singlet at 6.8 ppm. These peaks show an STD signal in the presence of MALAT1 triplex, suggesting that this part of the molecule is interacting with the RNA.