Profiling Cellular Morphological Changes Induced by Dual-targeting PROTACs of Aurora Kinase and RNA-binding Protein YTHDF2.

Proteolysis targeting chimeras (PROTACs) are new chemical modalities that degrade proteins of interest, including established kinase targets and emerging RNA-binding proteins (RBPs). Whereas diverse sets of biochemical, biophysical and cellular assays are available for the evaluation and optimizations of PROTACs in understanding the involved ubiquitin-proteasome-mediated degradation mechanism and the structure-degradation relationship, a phenotypic method profiling the cellular morphological changes is rarely used. In this study, first, we reported the only examples of PROTACs degrading the mRNA-binding protein YTHDF2 via screening of multikinase PROTACs. Second, we reported the profiling of cellular morphological changes of the dual kinase- and RBP-targeting PROTACs using the unbiased cell painting assay (CPA). The CPA analysis revealed the high biosimilarity with the established aurora kinase cluster and annotated aurora kinase inhibitors, which reflected the association between YTHDF2 and the aurora kinase signaling network. Broadly, the results demonstrated that the cell painting assay can be a straightforward and powerful approach to evaluate PROTACs. Complementary to the existing biochemical, biophysical and cellular assays, CPA provided a new perspective in characterizing PROTACs at the cellular morphology.

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.

Aminothiazolone Inhibitors Disrupt the Protein-RNA Interaction of METTL16 and Modulate the m6A RNA Modification.

Targeting RNA-binding and modifying proteins via small molecules to modulate post-transcriptional modifications have emerged as a new frontier for chemical biology and therapeutic research. One such RNA-binding protein that regulates the most prevalent eukaryotic RNA modification, N6-methyladenosine (m6A), is the methyltransferase-like protein 16 (METTL16), which plays an oncogenic role in cancers by cofunctioning with other nucleic acid-binding proteins. To date, no potent small-molecule inhibitor of METTL16 or modulator interfering with the METTL16-RNA interaction has been reported and validated, highlighting the unmet need to develop such small molecules to investigate the METTL16-involved regulatory network. Herein, we described the identification of a series of first-in-class aminothiazolone METTL16 inhibitors via a discovery pipeline that started with a fluorescence-polarization (FP)-based screening. Structural optimization of the initial hit yielded inhibitors, such as compound 45, that showed potent single-digit micromolar inhibition activity against the METTL16-RNA binding. The identified aminothiazolone inhibitors can be useful probes to elucidate the biological function of METTL16 upon perturbation and evaluate the therapeutic potential of METTL16 inhibition via small molecules at the post-transcriptional level.

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.

Kinase-targeting small-molecule inhibitors and emerging bifunctional molecules.

Kinases are among the most successful drug targets. To date, 72 small-molecule kinase inhibitors (SMKIs) have been approved by the US FDA, together with ~500 SMKIs in clinical trials. Although the topic has been heavily reviewed in recent years, an overview that focused on the currently approved SMKIs in combination with the emerging kinase-targeting bifunctional molecules is absent. Herein, we first provide an updated overview of the approved SMKIs, with an emphasis on their binding modes, classified in groups of type I and II ATP-competitive inhibitors, type III and IV allosteric inhibitors, and covalent inhibitors. We then highlight the novel chemical modalities in kinase targeting by using different types of proximity-inducing bifunctional molecules for kinase degradation and modifications.

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.

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.