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.

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.

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.

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.

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.

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.

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.

Discovery of selective CDK9 degraders with enhancing antiproliferative activity through PROTAC conversion.

Cyclin-dependent kinase 9 (CDK9) is an increasingly important potential cancer treatment target. Nowadays, developing selective CDK9 inhibitors has been extremely challenging as its ATP-binding sites are similar with other CDKs. Here, we report that the CDK9 inhibitor BAY-1143572 is converted into a series of proteolysis targeting chimeras (PROTACs) which leads to several compounds inducing the degradation of CDK9 in acute myeloid leukemia cells at a low nanomolar concentration. In addition, the most potent PROTAC molecule B03 could inhibit cell growth more effectively than warhead alone, with little inhibition of other kinases. This enhanced antiproliferative activity is mediated by a slight increase in kinase inhibitory activity and an increase in the level of apoptosis induction. Moreover, B03 could induce the degradation of CDK9 in vivo. Our work provides evidence that B03 represents a lead for further development and that CDK9 degradation is a potential valuable therapeutic strategy in acute myeloid leukemia.

Recent discovery of phosphoinositide 3-kinase γ inhibitors for the treatment of immune diseases and cancers.

Recently, PI3Kγ, a vital kinase, which involved in numerous intracellular signaling pathways, has been considered as a promising drug target for the treatment of immune diseases and certain cancers. Before the 21st century, few selective PI3Kγ inhibitors were discovered because no non-conserved structure in the ATP binding sites of PI3Kγ had been found. Since the discovery of the non-ATP binding pocket, the reported structures of potent and selective PI3Kγ inhibitors have become more diverse, and one compound (IPI549) has entered Phase I clinical trial. This review centers on a general overview of PI3Kγ inhibitors in clinical and preclinical as well as further therapeutic applications in human diseases.

Palladium(II)-Catalyzed Reaction of Lawsones and Propargyl Carbonates: Construction of 2,3-Furanonaphthoquinones and Evaluation as Potential Indoleamine 2,3-Dioxygenase Inhibitors.

An efficient reaction utilizing propargyl carbonates through Claisen rearrangement to synthesize furanonaphthoquinones is described. The remarkable transformation exhibits excellent functional group tolerance, affording the target furanonaphthoquinones in moderate to good yields (41-85%) under mild reaction conditions. Scaled-up preparation of the model product can make this reaction a method of choice for synthesis of furanonaphthoquinone derivatives. The resulting furanonaphthoquinones were evaluated as potential indoleamine 2,3-dioxygenase inhibitors in vitro.

Recent discovery of indoleamine-2,3-dioxygenase 1 inhibitors targeting cancer immunotherapy.

There has been great attention on indoleamine-2,3-dioxygenase 1 (IDO1) around cancer immunotherapy because of its role in enabling cancers to evade the immune system. The most recent spurt of high potent IDO1 inhibitors has been driven by the solution of the increased crystal structures of inhibitors with IDO1. Though the structural information of the active site of IDO1 obtained from the crystals are quite similar, the structures of reported potent inhibitors are quite different. Besides, while thousands of bioactive small molecule inhibitors of IDO1 exist, to date, only five compounds have entered clinical trials. In an effort to obtain more clinical drugs targeting IDO1, more comprehensive understanding of the active site of IDO1 and the structures of existing potent IDO1 inhibitors are necessary. Thus, this review mainly focus on the key features reported from specific crystal structures of IDO1 and an overview of the most recently developed IDO1 inhibitors under investigation and their other derived applications which may contribute to a better usage in cancer immunotherapy.

Discovery and synthesis of novel Wogonin derivatives with potent antitumor activity in vitro.

Phenotypic screening of high quality compound library is one of the most effective strategy to obtain novel bioactive compounds. Recently, our group have constructed a Wogonin-scaffold library with substituents diversity and successfully obtained a series of potent compounds. Herein, we reported the synthesis of these compounds and evaluated the in vitro antitumor activity against a panel of human tumor cell lines. Most of them showed good activity with a broad spectrum and preliminary structure-activity relationship for the substitutions were obtained. Further biological assays showed that the most potent compounds 18n and 20b could significantly enhance the intracellular ROS level and induce the cell apoptosis at low micromole level. Through similarity searching, CDK9 was identified as the potential target for 20b, which could be a start point for next structure-based drug design.