Chromenopyrazole-Peptide Conjugates as Small-Molecule Based Inhibitors Disrupting the Protein-RNA Interaction of LIN28-let-7.

Targeting the protein-RNA interaction of LIN28 and let-7 is a promising strategy for the development of novel anticancer therapeutics. However, a limited number of small-molecule inhibitors disrupting the LIN28-let-7 interaction with potent efficacy are available. Herein, we developed a novel LIN28-inhibiting strategy by targeting selective hotspot amino acids at the LIN28-let-7 binding interface with small-molecule-based bifunctional conjugates. Starting from reported small-molecule LIN28 inhibitors, we identified a feasible linker-attachment position after performing a structure-activity relationship exploration based on the LIN28-targeting chromenopyrazoles. In parallel, a virtual alanine scan identified hotspot residues at the protein-RNA binding interface, based on which we designed a set of peptides to enhance the interaction with the identified hotspot residues. Conjugation of the tailor-designed peptides with linker-attached chromenopyrazoles yielded a series of bifunctional small-molecule-peptide conjugates, represented by compound 83 (PH-223), as a new LIN28-targeting chemical modality. Our result demonstrated an unexplored rational design approach using bifunctional conjugates to target protein-RNA interactions.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-Binding Protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressing JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

Synthesis and evaluation of RNase L-binding 2-aminothiophenes as anticancer agents.

Aminothiophene is a scaffold that is widely present in drugs and biologically active small molecules as chemical probes. In this study, 43 compounds sharing a 2-aminothiophenone-3-carboxylate (ATPC) scaffold, known to activate the ribonuclease L (RNase L), were synthesized and selected ATPCs showed enhancement of thermal stability of RNase L upon binding. Screening of antiproliferation activities against human cancer cell lines revealed that ATPCs represented by compounds 4l and 50 showed potent single-digit micromolar antiproliferation activity against human cancer cell lines. Compounds 4l and 50 exhibited time- and dose-dependent proliferation inhibition, induced cellular apoptosis measured by cleaved PARP and via flow cytometry, inhibited cell migration, and inhibited cell colony formation. Combining the results reported in this work, ATPCs were evaluated as potential anticancer agents mediated by RNase L-binding and apoptosis induction. The work contributes to the study on the polypharmacological properties of aminothiophene-containing small molecules.

Targeting Ribonucleases with Small Molecules and Bifunctional Molecules.

Ribonucleases (RNases) cleave and process RNAs, thereby regulating the biogenesis, metabolism, and degradation of coding and noncoding RNAs. Thus, small molecules targeting RNases have the potential to perturb RNA biology, and RNases have been studied as therapeutic targets of antibiotics, antivirals, and agents for autoimmune diseases and cancers. Additionally, the recent advances in chemically induced proximity approaches have led to the discovery of bifunctional molecules that target RNases to achieve RNA degradation or inhibit RNA processing. Here, we summarize the efforts that have been made to discover small-molecule inhibitors and activators targeting bacterial, viral, and human RNases. We also highlight the emerging examples of RNase-targeting bifunctional molecules and discuss the trends in developing such molecules for both biological and therapeutic applications.

Rational design and evaluation of 2-((pyrrol-2-yl)methylene)thiophen-4-ones as RNase L inhibitors.

Ribonuclease L (RNase L) plays a crucial role in an antiviral pathway of interferon-induced innate immunity by degrading RNAs to prevent viral replication. Modulating RNase L activity thus mediates the innate immune responses and inflammation. Although a few small molecule-based RNase L modulators have been reported, only limited molecules have been mechanistically investigated. This study explored the strategy of RNase L targeting by using a structure-based rational design approach and evaluated the RNase L-binding and inhibitory activities of the yielded 2-((pyrrol-2-yl)methylene)thiophen-4-ones, which exhibited improved inhibitory effect as determined by in vitro FRET and gel-based RNA cleavage assay. A further structural optimization study yielded selected thiophenones that showed >30-fold more potent inhibitory activity than that of sunitinib, the approved kinase inhibitor with reported RNase L inhibitory activity. The binding mode with RNase L for the resulting thiophenones was analyzed by using docking analysis. Furthermore, the obtained 2-((pyrrol-2-yl)methylene)thiophen-4-ones exhibited efficient inhibition of RNA degradation in cellular rRNA cleavage assay. The newly designed thiophenones are the most potent synthetic RNase L inhibitors reported to date and the results revealed in our study lay the foundation for the development of future RNase L-modulating small molecules with new scaffold and improved potency.

N-Biphenyl Pyrrolinones and Dibenzofurans as RNA-Binding Protein LIN28 Inhibitors Disrupting the LIN28-Let-7 Interaction.

The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.

Small molecules with tetrahydroquinoline-containing Povarov scaffolds as inhibitors disrupting the Protein-RNA interaction of LIN28-let-7.

Inhibition of the RNA-binding protein LIN28 and disruption of the protein-RNA interaction of LIN28-let-7 with small molecules holds great potential to develop new anticancer therapeutics. Herein, we report the LIN28 inhibitory activities of a series of 30 small molecules with a tricyclic tetrahydroquinoline (THQ)-containing scaffold obtained from a Povarov reaction. The THQ molecules were structurally optimized by varying the 2-benzoic acid substituent, the fused ring at 3- and 4-positions, and the substituents at the phenyl moiety of the tetrahydroquinoline core. Among the tested compounds, GG-43 showed dose-dependent inhibition in an EMSA validation assay and low micromolar inhibitory activity in a fluorescence polarization-based assay measuring disruption of LIN28-let-7 interaction. Binding mode between GG-43 and the cold shock domain of LIN28 was proposed via a molecular docking analysis. The study provides one of the first systematic analyses on structural features that are required for LIN28 inhibition, and indicates the necessity to develop small molecules with new scaffolds as LIN28-targeting probes and therapeutic candidates. In parallel, this study demonstrates the polypharmacological nature of tricyclic THQ-containing scaffolds accessible through Povarov reactions.

Small-molecule screening of ribonuclease L binders for RNA degradation.

Small molecules targeting the ubiquitous latent ribonuclease (RNase L), which has limited sequence specificity toward single-stranded RNA substrates, hold great potential to be developed as broad-spectrum antiviral drugs by modulating the RNase L-mediated innate immune responses. The recent development of proximity-inducing bifunctional molecules, as described in the strategy of ribonuclease targeting chimeras, demonstrated that small-molecule RNase L activators can function as the essential RNase L-recruiting component to design bifunctional molecules for targeted RNA degradation. However, only a single screening study on small-molecule RNase L activators with poor potency has been reported to date. Herein, we established a FRET assay and conducted a screening of 240,000 small molecules to identify new RNase L activators with improved potency. The extremely low hit rate of less than 0.03% demonstrated the challenging nature of RNase L activation by small molecules available from current screening collections. A few hit compounds induced enhanced thermal stability of RNase L upon binding, although validation assays did not lead to the identification of compounds with significantly improved RNase L activating potency. The sulfonamide compound 17 induced a thermal shift of ~ 0.9 °C upon binding to RNase L, induced significant apoptosis in cancer cells, and showed single-digit micromolar inhibitory activity against cancer cell proliferation. This study paves the way for future structural optimization for the development of small-molecule RNase L binders.

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.

Multicomponent Petasis Reaction for the Synthesis of Functionalized 2-Aminothiophenes and Thienodiazepines.

Multicomponent Petasis reaction has been widely applied for the synthesis of functionalized amine building blocks and biologically active compounds. Employing primary aromatic amines that are not typical reactive substrates contributes to expand the application scope of the Petasis reaction. In this study, we demonstrated the synthesis of functionalized 2-aminothiophenes using Gewald-reaction-derived 2-aminothiophenes as the amine substrates, whose low reactivity in the Petasis reaction was overcome using hexafluoro-2-propanol as the solvent in a mild condition. The obtained Petasis products are amenable for further transformations owing to the presence of multiple functional handles. A following intramolecular cyclization of selected Petasis products afforded substituted tricyclic heterocycles that incorporate a pharmaceutically interesting thienodiazepine moiety.