Trisubstituted Pyrrolinones as Small-Molecule Inhibitors Disrupting the Protein–RNA Interaction of LIN28 and Let-7

Modulation of protein–RNA interaction (PRI) using small molecules is a promising strategy to develop therapeutics. LIN28 is an RNA-binding protein that blocks the maturation of the tumor suppressor let-7 microRNAs. Herein, we performed a fluorescence polarization-based screening and identified trisubstituted pyrrolinones as small-molecule inhibitors disrupting the LIN28–let-7 interaction. The most potent compound C902 showed dose-dependent inhibition in an EMSA validation assay, enhanced thermal stability of the cold shock domain of LIN28, and increased mature let-7 levels in JAR cells. The structure–activity relationship study revealed key structural features contributing to either PRI inhibition or stabilization of protein–protein interaction (PPI). The pyrrolinones identified in this study not only represent a new class of LIN28-binding molecules that diversify the limited available LIN28 inhibitors but also represent the first examples of small molecules that showed substituent-dependent PRI inhibitory and PPI activating activities.

S4 was detected after incubation at room temperature using a TECAN Spark plate reader. Half maximal inhibitory concentrations (IC50) were determined using GraphPad Prism 7.
Compound screening. High throughput screening was performed in low volume 384 well plates (Corning 4514) with 30 µM compound, 40 nM LIN28A, and 2 nM preE-let-7f-1-FAM in FP assay buffer. Compounds were transferred with an ECHO 520 liquid handler and protein and RNA were added with a multidrop dispenser. Protein and RNA were added stepwise with a 30 min incubation period in between. FP was detected after a final incubation of 15 min with an Envision plate reader and fluorescence was measured with a Paradigm plate reader. A reaction without LIN28 served as a negative control and one without compound was used as a positive control. The software Quattro Workflow (Quattro Research GmbH) was used for the analysis of screening data.
Electrophoretic mobility shift assay. EMSA was used as a secondary assay to validate FP-assay hit molecules. Purified LIN28A (residues 16-187) was incubated with compound and 5 U recombinant ribonuclease inhibitor (Takara Bio) in EMSA reaction buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 10 mM β-mercaptoethanol, 50 µM ZnCl2, 2 % DMSO, 0.01 % Tween 20, 12 % glycerol) for 2 hours at room temperature. Subsequently, preE-let-7f-1-Cy3 (mus musculus) (GGGGUAGUGAUUUUAC CCUGUUUAGGAGAU-Cy3, purchased from IDT) was added to a final concentration of 5 nM and a reaction volume of 50 µL. The final concentration of LIN28A was 10 nM and compound concentrations up to 75 µM were used. The reaction mixtures were incubated for a further 15 minutes and 10 µL loading dye (40 % glycerol, 1.5x TAE) was added. 10 µL of each reaction was separated in an 8 % polyacrylamide TAE gel at 4 °C and 220 V for 1 h using 0.25x TAE as running buffer. Cy3 fluorescence was detected with a ChemiDoc MP (Bio-Rad) and 2 minutes of exposure time.
Thermal shift assay. Thermal stability of the cold shock domain construct LIN28A (residues 16-126) with and without compound was monitored using a NanoTemper Prometheus NT.48 nano-S5 differential scanning fluorimetry (nanoDSF) instrument at temperatures between 20 °C and 90 °C and a temperature slope of 1 °C per minute with an excitation power of 50-60 %. A total protein concentration of 30 µM and compound concentrations from 100 µM to 0.78 µM corresponding to 5 % to 0.039 % DMSO, respectively, were used. Compound and protein were incubated for 45 minutes at room temperature in buffer containing 30 mM NaH2PO4, 50 mM NaCl, 1 mM MgCl2, and 50 µM ZnCl2 at pH 8 and 10 µL sample was loaded to each nanoDSF standard capillary (NanoTemper). The ratio of intrinsic tryptophan fluorescence at 350 nm and 330 nm was measured and the first derivative was determined to detect the inflection points using the software of the device.

RT-qPCR. JAR cells (obtained from DSMZ, German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany, DSMZ no. ACC462) 1 were cultured in 24-well plates and treated with the compound (PH-31) for 24 h with a total DMSO concentration of 0.5 % in all wells. RNA was extracted using RNeasy Mini Kit (Qiagen) and 20 ng of total RNA was used for reverse transcription using the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's protocols. We then performed qPCR using TaqMan Universal Master Mix II, with UNG (Applied Biosystems) according to the manufacturer's protocol using a CFX Connect Real-Time PCR System (BioRad). TaqMan microRNA assays were purchased from Applied Biosystems (Assay IDs: 001093, 000377, 002282). Relative expression levels were normalized to U6 snRNA and samples treated with DMSO using the reported 2 -ΔΔCT method. 2 Docking analysis. Computational docking of pyrrolinones to preE-let-7 binding site of LIN28 CSD (PDB code: 5UDZ) was done by Schrödinger Maestro 12.3. The 3-dimensional structures of pyrrolinones were prepared after calculating energy minimization by mm2 at Chem3D 18.2 and the chemical states were generated by the ligand preparation module. The conformation of LIN28 was prepared according to the protein preparation module, including hydrogen addition, water molecule removal, and energy minimization, after removing preE-let-7 fragments except for oligomers used as S6 ligand. The binding site was generated by grid generation module and using preE-let-7 fragment (A6-U8, U8-U11, or U13-A15) as the ligand. The glide dock was performed by glide dock module and evaluated according to the docking score, small molecule orientation, solvent exposure, and interactions between small molecules and LIN28. The interactions were visualized by using PyMOL.   Table 1 of the main manuscript. S12 Figure S7. Comparison of the LIN28-let-7 inhibitory activity and 14-3-3-PMA2 stabilizing activity of representative 1,4,5-trisubstituted pyrrolinones and 3,4,5-trisubstituted pyrazoles. Pyrrolinones showed micromolar LIN28-let-7 inhibitory activity and minimal 14-3-3-PMA2 stabilizing activity, while pyrazoles are inactive against LIN28-let-7 but are favored scaffolds for 14-3-3-PMA2 stabilization. The IC50 against LIN28-let-7f-1 are shown in green rectangles and the EC50 towards the 14-3-3 protein T14-3e and the PMA2-CT52YDI protein are shown in blue rectangles (including stabilizing effect in % towards T14-3c-CT66YDI protein at a compound concentration of 100 µM). 3 PPI, protein-protein interaction; PRI, protein-RNA interaction. S13 Table S1. LIN28-let-7 inhibitory activity of trisubstituted pyrazoles   Table 1 and Table S1)        High-resolution mass spectrometry (HRMS) was measured on an LTQ Orbitrap mass spectrometer coupled to an Accela HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1 mm, particle size 1.9 μm, ionization method: electron spray ionization (ESI)).

General step for the Doebner condensation
The benzaldehyde derivative (1.0 equiv.) and the aniline derivative (1.0 equiv.) were added sequentially to a suspension (0.06 M) of the dioxobutanoate component (1.0 equiv.) in acetic acid. 3 The reaction mixture was stirred overnight at 90 °C. After cooled to room temperature, the reaction mixture was diluted with Et2O and filtered. The residue was washed with Et2O to give the desired pyrrolinone derivative after drying. In the case that precipitation did not yield pure product, the precipitate was further purified with an appropriate gradient on a preparative HPLC system.