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.

Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

Discovery of a Drug-like, Natural Product-Inspired DCAF11 Ligand Chemotype.

Targeted proteasomal and autophagic protein degradation, often employing bifunctional modalities, is a new paradigm for modulation of protein function. In an attempt to explore protein degradation by means of autophagy we combine arylidene-indolinones reported to bind the autophagy-related LC3B-protein and ligands of the PDEδ lipoprotein chaperone, the BRD2/3/4-bromodomain containing proteins and the BTK- and BLK kinases. Unexpectedly, the resulting bifunctional degraders do not induce protein degradation by means of macroautophagy, but instead direct their targets to the ubiquitin-proteasome system. Target and mechanism identification reveal that the arylidene-indolinones covalently bind DCAF11, a substrate receptor in the CUL4A/B-RBX1-DDB1-DCAF11 E3 ligase. The tempered α, ß-unsaturated indolinone electrophiles define a drug-like DCAF11-ligand class that enables exploration of this E3 ligase in chemical biology and medicinal chemistry programs. The arylidene-indolinone scaffold frequently occurs in natural products which raises the question whether E3 ligand classes can be found more widely among natural products and related compounds.

Morphological subprofile analysis for bioactivity annotation of small molecules.

Fast prediction of the mode of action (MoA) for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. Morphological profiling, e.g., using the Cell Painting assay, offers a fast, unbiased assessment of compound activity on various targets in one experiment. However, due to incomplete bioactivity annotation and unknown activities of reference compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the MoA for both, reference and unexplored compounds. We defined MoA clusters and extracted cluster subprofiles that contain only a subset of morphological features. Subprofile analysis allows for the assignment of compounds to, currently, twelve targets or MoA. This approach enables rapid bioactivity annotation of compounds and will be extended to further clusters in the future.

Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

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.

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.

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.

Discovery of Dihydro-1,4-Benzoxazine Carboxamides as Potent and Highly Selective Inhibitors of Sirtuin-1.

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.

Cell-Based Identification of New IDO1 Modulator Chemotypes.

The immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO1) strengthens cancer immune escape, and inhibition of IDO1 by means of new chemotypes and mechanisms of action is considered a promising opportunity for IDO1 inhibitor discovery. IDO1 is a cofactor-binding, redox-sensitive protein, which calls for monitoring of IDO1 activity in its native cellular environment. We developed a new, robust fluorescence-based assay amenable to high throughput, which detects kynurenine in cells. Screening of a ca. 150 000-member compound library discovered unprecedented, potent IDO1 modulators with different mechanisms of action, including direct IDO1 inhibitors, regulators of IDO1 expression, and inhibitors of heme synthesis. Three IDO1-modulator chemotypes were identified that bind to apo-IDO1 and compete with the heme cofactor. Our new cell-based technology opens up novel opportunities for medicinal chemistry programs in immuno-oncology.

PARL partitions the lipid transfer protein STARD7 between the cytosol and mitochondria.

Intramembrane-cleaving peptidases of the rhomboid family regulate diverse cellular processes that are critical for development and cell survival. The function of the rhomboid protease PARL in the mitochondrial inner membrane has been linked to mitophagy and apoptosis, but other regulatory functions are likely to exist. Here, we identify the START domain-containing protein STARD7 as an intramitochondrial lipid transfer protein for phosphatidylcholine. We demonstrate that PARL-mediated cleavage during mitochondrial import partitions STARD7 to the cytosol and the mitochondrial intermembrane space. Negatively charged amino acids in STARD7 serve as a sorting signal allowing mitochondrial release of mature STARD7 upon cleavage by PARL On the other hand, membrane insertion of STARD7 mediated by the TIM23 complex promotes mitochondrial localization of mature STARD7. Mitochondrial STARD7 is necessary and sufficient for the accumulation of phosphatidylcholine in the inner membrane and for the maintenance of respiration and cristae morphogenesis. Thus, PARL preserves mitochondrial membrane homeostasis via STARD7 processing and is emerging as a critical regulator of protein localization between mitochondria and the cytosol.

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.

Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics.

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin-like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1-mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress-sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1-mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.