Articles pubs.acs.org/acschemicalbiology
A Small-Molecule Inhibitor of Lin28 Martina Roos,†,⊥ Ugo Pradère,†,⊥ Richard P. Ngondo,‡ Alok Behera,† Sara Allegrini,§ Gianluca Civenni,§ Julian A. Zagalak,† Jean-Rémy Marchand,∥ Mirjam Menzi,† Harry Towbin,† Jörg Scheuermann,† Dario Neri,† Amedeo Caflisch,∥ Carlo V. Catapano,§ Constance Ciaudo,‡ and Jonathan Hall*,† †
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, 8093 Zurich, Switzerland § Institute of Oncology Research, Oncology Institute of Southern Switzerland, 6500 Bellinzona, Switzerland ∥ Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland ‡
S Supporting Information *
ABSTRACT: New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs. Here, we describe a protein/RNA FRET assay using a GFP-Lin28 donor and a black-hole quencher (BHQ)-labeled let-7 acceptor, a fluorescent protein/quencher combination which is rarely used in screening despite favorable spectral properties. We tested 16 000 molecules and identified N-methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide, which blocked the Lin28/let-7 interaction, rescued let-7 processing and function in Lin28-expressing cancer cells, induced differentiation of mouse embryonic stem cells, and reduced tumor-sphere formation by 22Rv1 and Huh7 cells. A biotinylated derivative captured Lin28 from cell lysates consistent with an on-target mechanism in cells, though the compound also showed some activity against bromodomains in selectivity assays. The Lin28/let-7 axis is presently of high interest not only for its role as a bistable switch in stem-cell biology but also because of its prominent roles in numerous diseases. We anticipate that much can be learned from the use of this first reported small molecule antagonist of Lin28, including the potential of the Lin28/let-7 interaction as a new drug target for selected cancers. Furthermore, this approach to assay development may be used to identify antagonists of other RBP/RNA interactions suspected to be operative in pathophysiological mechanisms.
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their processing by Drosha and Dicer7−15 (Figure 1a). Let-7 controls cell proliferation, and its targets include the mRNAs of important oncogenes such as K-RAS, MYC, and LIN28.16−18 Multiple lines of evidence suggest that the Lin28/let-7 relationship plays a prominent role in cancer. For example, low levels of let-7 in several cancers are associated with a poor prognosis;19 increased expression of let-7 levels inhibit tumor growth in mouse models.20 Lin28 overexpression in mice confers tumorigenic properties, whereas its inhibition decreases cancer cell survival.21 These observations suggest that the Lin28/let-7 interaction might be an attractive target for conventional therapeutics; however, the well-known difficulties in targeting RNA-protein interactions22,23 with small-molecules hamper validation of this hypothesis.
icroRNAs (miRNAs) are small RNAs which suppress gene expression post-transcriptionally.1 Their biogenesis passes through two precursor intermediates: a primary miRNA transcript (pri-miRNA) containing a stem-loop structure which is cleaved by the nuclear RNase III Drosha2 and the premiRNA from which the terminal loop region (TLR) is cleaved by the cytoplasmic RNase III Dicer (Figure 1a).3,4 One arm of the remaining double-stranded RNAthe mature miRNAis then taken into the miRNA-induced silencing complex (miRISC) with an Argonaute (Ago) protein. MiRISC complexes bind to the 3′ untranslated regions (3′UTRs) of mRNAs and suppress gene expression.1 In mammals, the precursors of the let-7 family are abundant in embryonic stem cells (ESCs), but mature let-7 only appears at later developmental stages.5 In humans, 10 let-7’s are expressed from 13 distinct precursors.6 Their maturation is controlled by Lin28, a small RBP expressed in ESCs, with important roles in development and disease. Humans express two isoforms of Lin28, LIN28 (Lin28A) and LIN28B (Lin28B), which bind to conserved sites present in let-7 precursors and thereby inhibit © XXXX American Chemical Society
Received: March 11, 2016 Accepted: August 2, 2016
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DOI: 10.1021/acschembio.6b00232 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Articles
ACS Chemical Biology
provided us the key flexibility needed to change the position, as well as the number and the nature of the acceptors on the premiRNA in order to maximize the FRET (Figure 1b). The acceptor chromophores were introduced after synthesis of the fully protected oligoribonucleotide by a Cu(I)-mediated cycloaddition of azide-functionalized acceptor probes at cytidines and adenosines bearing a 2′-O-propargyl substituent (Figure 2a, Table S1). The procedure yielded labeled premiRNAs of high purity (Figure S1; Table S1).
Figure 1. Biogenesis of miRNAs. (a) Pri-let-7 is processed to mature let-7 by Drosha and Dicer; maturation is blocked by Lin28 binding to the TLs of let-7 precursors. (b) N-terminal-EGFP-tagged-Lin28 binding to pre-let-7a-2 (Figure S9): positions of FRET acceptor conjugation are numbered.
One method to identify inhibitors of bimolecular interactions is compound screening. Fluorescence resonance energy transfer (FRET)-based assays are well-established for protein−protein interactions, where interacting partners are linked to donor and acceptor protein fluorophores. They have also been used for screening protein−RNA interactions, where fluorophores are linked to the interacting partners through proteins or antibodies.24 However, we considered that it would be advantageous if the RNA could be labeled directly with a discrete chromophore. This would enable optimization of donor−acceptor distance/orientation in order to maximize FRET efficiency, which is critical for assay miniaturization. Hence, a flexible RNA-labeling strategy and a judicious choice of donor−acceptor pairs are key elements in screen design, for which there are few literature precedents. Here, we describe a novel screening assay comprising Lin28 and labeled let-7, made possible by state-of-the-art RNA chemistry. We screened 16 000 drug-like molecules and identified and characterized one with on-target micromolar activity in Lin-28-expressing murine ESCs (mESCs) and liver cancer cell lines. We anticipate that this chemical tool compound will be used to expand our knowledge of the Lin28/let-7 axis in (cancer) stem cells and the potential of this protein/RNA interaction as a new target for certain cancers. The introduction of such assay formats opens access to compounds to investigate increasing numbers of newly discovered RBP/noncoding RNA interactions.
Figure 2. (a) Cy3 or BHQ-1 chromophores conjugated to propargylated adenosine or cytosine residues (left); LC-MS chromatogram of 19B-let7 (right). (b) Effects of graded concentrations of Cy3labeled pre-miRNAs on EGFP signal intensity (FRET). 10Cy3-let7/ let7 refers to equimolar mixtures of 10Cy3-let7 and unlabeled pre-let7a-2. (c) FRET with various Cy3- or BHQ-1-labeled pre-let-7a-2’s at 5 nM (positions illustrated in Figure 1b). (d) Effects of graded concentrations of BHQ-1-labeled pre-miRNAs on FRET. Error bars indicate ±1 SD (n = 2).
We labeled truncated pre-let-7a-2one family member of the let-7 precursors (see Supporting Information)at its 5′position with Cy3 (10Cy3-let7) and evaluated its performance in the FRET assay (Figure 2b). As a control to indicate possible unspecific protein−RNA binding, we labeled pre-miR-32, which we have shown previously binds very weakly to Lin2826 (1Cy3-miR32: Table S1). At a concentration of 20 nM, 10Cy3-let7 induced a FRET of 13%, whereas 1Cy3-miR32 was ineffective (Figure 2b). The addition of an equimolar amount of nonlabeled pre-let-7a-2 to the 10Cy3-let7-containing solution reduced the FRET by approximately 2-fold, suggesting that Lin28 bound the labeled and wild-type RNAs similarly and therefore that Lin28 binding was probably not affected adversely by the Cy3 fragment. We considered a FRET of 13% too small for assay miniaturization and compound screening. Therefore, we attempted to increase the FRET efficiency by relocating the Cy3 probe to alternative positions in the stem-loop and chose position 19 in the 5p arm (19Cy3let7), position 34 in the TLR (34Cy3-let7), and position 57 (57Cy3-let7) corresponding to the 3′-end of the truncated premiRNA (Figure 1b, Table S1). Compared under identical conditions to 10Cy3-let7 with 13% FRET, all three RNAs showed increased efficiencies: 57Cy3-let7 (16%), 19Cy3-let7 (21%), and 34Cy3-let7 (18%) (Figure 2c). We attributed these improvements to a closer distance between the donor and the acceptor, though they might also have resulted from favorable changes in the relative orientation of the chromophores’ dipole moment.27 Although the improvements demonstrated the value
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RESULTS AND DISCUSSION Development of an Assay Sensor. We initially chose enhanced green fluorescent protein (EGFP)-cyanine 3 dye (Cy3) as the donor−acceptor combination, because it is one of the most commonly used fluorescent-protein/organic-chromophore FRET pairs. We prepared an N-terminal EGFP-tagged Lin28 sequence by amplification of Lin28B cDNA and cloned it into a mammalian expression vector. This vector was used to generate a HEK 293T cell line stably expressing a constant level of EGFP-Lin28B protein from which lysate batches were harvested for FRET experiments. We anticipated that the sensitivity of the sensor would be strongly influenced by the position of the FRET acceptor on the structured RNA. Therefore, we employed a multisite specific labeling technique which we have recently developed for pre-miRNAs.25 This B
DOI: 10.1021/acschembio.6b00232 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Articles
ACS Chemical Biology
Figure 3. Structures and activities of selected hits. (a) 14 hits identified for follow-up. (b) Let-7 activity of selected hits in luciferase reporter gene assays relative to DMSO. (c) Cellular levels of mature let-7a, -7g, -7f, and mir-15 relative to DMSO, 48 h after treatment (n = 2). (d) Inhibition by 1632 of c-Myc-Lin28A binding to pre-let-7a-2. (e) Capture of proteins from HeLa cell lysates transfected with c-Myc-Lin28A expression plasmid using 1632Bio. Error bars indicate ±1 SD (n = 3) in b, d, and e. *P < 0.05; **P < 0.01.
of a versatile RNA conjugation chemistry for fine-tuning the intensity of the FRET, we pursued the introduction of multiple FRET acceptors for further improvements. Hence, we added a second Cy3 fragment to the pre-let-7a-2 in an effort to enhance the FRET, bis-labeling simultaneously at positions 10 and 19 (10−19Cy3-let7) or 19 and 34 (19−34Cy3-let7; Table S1). We also bis-labeled pre-miR101 as a second negative control26 in positions 1 and 8 (1−8Cy3-miR101). Bis-Cy3-labeled pre-let7’s yielded strong FRET effects of 29% and 33% already at low RNA concentrations of 5 nM (Figure 2c) while negative control 1−8Cy3-miR101 was unresponsive (Figure S2). This increase of FRET was however accompanied by increased spectral bleed-through, which lowered assay sensitivity. As we favored measurement of FRET via decreased donor emission, we turned to the use of a quencher moleculeblack-holequencher 1 (BHQ-1) (Figure 2a)as the acceptor which absorbs from 400 to 650 nm but does not re-emit in the visible range (Figure S3). This combination of fluorescent protein/ small molecule quencher is rarely used for screening, despite its highly advantageous donor−acceptor spectral overlaps for FRET applications.28 In a similar procedure, we mono- and bis-labeled truncated pre-let-7a-2 with BHQ-1 at positions 10, 19, and 34, as well as a negative control pre-miR-101 on its 5′end (1B-miR101; Table S1). The control showed no FRET at an RNA concentration of 5 nM (Figure S2), whereas pre-let-7a2 labeled with BHQ-1 at positions 10, 19, and 34 showed strong FRET effects (23%, 33%, and 24%, respectively; Figures 2c,d). Bis-BHQ-1-labeled RNA (10−19B-let7) did not yield higher FRET than the singly labeled 19B-let7 (Figure 2c). Taking these observations together, we therefore opted to use 19B-let7 for the compound screen. Compound Screen for Inhibitors of pre-let-7/Lin28 Binding. We tested a library of 16 000 small drug-like molecules (Maybridge Hitfinder library) in order to identify compounds that inhibit the interaction between EGFP-tagged Lin28B and 19B-let7, as revealed by a reduction of the FRET.
For a positive control compound, we used a 2′-O-methyloligoribonucleotide (L29−13) which we showed recently prevents Lin28 from binding to the pre-let-7a-2 loop.29 L29− 13 almost fully attenuated the FRET (vide inf ra). We employed the strictly standardized mean difference (SSMD*) method for analysis of the screening data. We carried out a pilot experiment in which microtiter wells containing EGFP-Lin28/19B-let7/ L29−13 produced an SSMD* = 2.92 compared to wells devoid of the oligonucleotide (Figure S4a). According to recently published recommendations, these values suggested the assay was of good quality and would permit clear identification of hits.30 The majority of the tested compounds had only low or no effects in the assay (Figure S4b). Hit selection was set by lower and upper SSMD* thresholds which corresponded roughly to 66% and 133% of the baseline reference, respectively. This cutoff highlighted 203 hits which were then re-evaluated in triplicate in a new screen (Figure S5a), correcting for compound self-fluorescence to remove false positives. Using unpaired t-statistics, 14 compounds from the 203 compounds were reserved for follow-up studies (Figure 3a; Figure S5b). These hit compounds are low molecular weight (
