Structure-activity relationship study of small-molecule inhibitor of Atg12-Atg3 protein-protein interaction.

Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.

High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified hits that inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Counter-screening against pseudoviruses with the vesicular stomatitis virus glycoprotein (VSV-G), yielded sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds, which included the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested against pseudoviruses bearing the S glycoprotein of the Alpha, Delta, and Omicron variants as well as bona fide SARS-CoV-2. This rigorous approach revealed an unreported inhibitor and its derivative as potential broad-spectrum antivirals.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 µM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1ß (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Benzylic Dehydroxylation of Echinocandin Antifungal Drugs Restores Efficacy against Resistance Conferred by Mutated Glucan Synthase.

Each year, infections caused by fungal pathogens claim the lives of about 1.6 million people and affect the health of over a billion people worldwide. Among the most recently developed antifungal drugs are the echinocandins, which noncompetitively inhibit ß-glucan synthase, a membrane-bound protein complex that catalyzes the formation of the main polysaccharide component of the fungal cell wall. Resistance to echinocandins is conferred by mutations in FKS genes, which encode the catalytic subunit of the ß-glucan synthase complex. Here, we report that selective removal of the benzylic alcohol of the nonproteinogenic amino acid 3S,4S-dihydroxy-l-homotyrosine of the echinocandins anidulafungin and rezafungin, restored their efficacy against a large panel of echinocandin-resistant Candida strains. The dehydroxylated compounds did not significantly affect the viability of human-derived cell culture lines. An analysis of the efficacy of the dehydroxylated echinocandins against resistant Candida strains, which contain mutations in the FKS1 and/or FKS2 genes of the parental strains, identified amino acids of the Fks proteins that are likely to reside in proximity to the l-homotyrosine residue of the bound drug. This study describes the first example of a chemical modification strategy to restore the efficacy of echinocandin drugs, which have a critical place in the arsenal of antifungal drugs, against resistant fungal pathogens.

PRMT1 inhibition induces differentiation of colon cancer cells.

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

Ubiquitous selection for mecA in community-associated MRSA across diverse chemical environments.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is threatening public health as it spreads worldwide across diverse environments. Its genetic hallmark, the mecA gene, confers resistance to many ß-lactam antibiotics. Here, we show that, in addition, mecA provides a broad selective advantage across diverse chemical environments. Competing fluorescently labelled wild-type and mecA-deleted CA-MRSA USA400 strains across ~57,000 compounds supplemented with subinhibitory levels of the ß-lactam drug cefoxitin, we find that mecA provides a widespread advantage across ß-lactam and non ß-lactam antibiotics, non-antibiotic drugs and even diverse natural and synthetic compounds. This advantage depends on the presence of cefoxitin and is strongly associated with the compounds' physicochemical properties, suggesting that it may be mediated by differential compounds permeability into the cell. Indeed, mecA protects the bacteria against increased cell-envelope permeability under subinhibitory cefoxitin treatment. Our findings suggest that CA-MRSA success might be driven by a cell-envelope mediated selective advantage across diverse chemical compounds.

MitoTimer-based high-content screen identifies two chemically-related benzothiophene derivatives that enhance basal mitophagy.

Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a 'basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.

In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.

Phenotypic Screen Identifies JAK2 as a Major Regulator of FAT10 Expression.

FAT10 is a ubiquitin-like protein suggested to target proteins for proteasomal degradation. It is highly upregulated upon pro-inflammatory cytokines, namely, TNFα, IFNγ, and IL6, and was found to be highly expressed in various epithelial cancers. Evidence suggests that FAT10 is involved in cancer development and may have a pro-tumorigenic role. However, its biological role is still unclear, as well as its biochemical and cellular regulation. To identify pathways underlying FAT10 expression in the context of pro-inflammatory stimulation, which characterizes the cancerous environment, we implemented a phenotypic transcriptional reporter screen with a library of annotated compounds. We identified AZ960, a potent JAK2 inhibitor, which significantly downregulates FAT10 under pro-inflammatory cytokines induction, in an NFκB-independent manner. We validated JAK2 as a major regulator of FAT10 expression via knockdown, and we suggest that the transcriptional effects are mediated through pSTAT1/3/5. Overall, we have elucidated a pathway regulating FAT10 transcription and discovered a tool compound to chemically downregulate FAT10 expression, and to further study its biology.

The small molecule Chicago Sky Blue promotes heart repair following myocardial infarction in mice.

The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria.

microRNAs (miRNAs) are critical for neuronal function and their dysregulation is repeatedly observed in neurodegenerative diseases. Here, we implemented high content image analysis for investigating the impact of several miRNAs in mouse primary motor neurons. This survey directed our attention to the neuron-specific miR-124, which controls axonal morphology. By performing next generation sequencing analysis and molecular studies, we characterized novel roles for miR-124 in control of mitochondria localization and function. We further demonstrated that the intermediate filament Vimentin is a key target of miR-124 in this system. Our data establishes a new pathway for control of mitochondria function in motor neurons, revealing the value of a neuron-specific miRNA gene as a mechanism for the re-shaping of otherwise ubiquitously-expressed intermediate filament network, upstream of mitochondria activity and cellular metabolism.

Notch Activation by Shootin1 Opposing Activities on 2 Ubiquitin Ligases.

The evolutionarily conserved Notch pathway plays an important role in regulation of stem cell renewal and cell fate determination in numerous organs, and as such is a key pathway in normal health and disease processes. Canonical Notch signaling is usually activated by cell contact where transmembrane ligands such as Delta-like and Jagged bind to Notch receptors. Notch activation results in the translocation of the cleaved Notch intracellular domain (NICD) into the nucleus and subsequent activation of transcription. Poly-ubiquitination leading to proteosome degradation of pathway components is one mean of regulating the Notch pathway. Here, we identified that Shootin1 exhibits the surprising propensity of activating the pathway either by interacting with LNX1/2 and promoting poly-ubiquitination of Numb or by complexing with Itch and impairing poly-ubiquitination of NICD. Within the developing brain Shootin1 modulates neuroblasts cell fate by executing 2 opposing activities on ubiquitin ligases, which control Notch signaling on 2 different levels.

A multiplexed screening method for pluripotency.

Measurement of Alkaline Phosphatase (ALP) level is a widely used procedure in clinical and basic research. We present a simple and inexpensive luminescence-based method that allows multiplexed measurement and normalization of intracellular ALP levels in one sample well. The method comprises two commercially available reagents enabling quantification of ALP levels and cell number by two sequential luminescence readouts. Using this method we were able to detect and analyze somatic reprogramming into pluripotent stem cells. The method is highly applicable for High Throughput Screening (HTS) campaigns and analysis.

Analysis of complex anisotropy decays from single-frequency polarized-phasor ellipse plots.

The anisotropy decay of a fluorescently-labelled macromolecule provides information on the internal and global dynamics of the macromolecule. Weber was a pioneer of fluorescent probes, polarization and polarized phase-modulation methods and revealed the power of combining or comparing these methods to disentangle complex modes of emission depolarization. In this paper we take a similar course and show that when measurements of dynamic depolarization are combined with steady-state anisotropy, complex anisotropy decays can be deduced from measurements at a single modulation frequency. Specifically, a double exponential anisotropy decay can be resolved by combining one of the polarized emission phasors with the steady-state anisotropy. The key is the polarized phasor ellipse plot which provides a convenient visualisation aid and reduces the dimensionality of the minimisation problem from three variables to one variable. We illustrate these concepts with an experimental measurement of the anisotropy decay of a small cytoplasmic fluorescent probe in live cells.

The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.

The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.

The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.

Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

Recruitment of the adaptor protein Grb2 to EGFR tetramers.

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM-FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2-mRFP) and EGFR (EGFR-eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2-mRFP with EGFR-eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR-eGFP per cluster. In contrast, the pool of EGFR-eGFP without Grb2-mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR-eGFP and Grb2-mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.

Exploring higher-order EGFR oligomerisation and phosphorylation--a combined experimental and theoretical approach.

The epidermal growth factor receptor (EGFR) kinase is generally considered to be activated by either ligand-induced dimerisation or a ligand-induced conformational change within pre-formed dimers. Ligand-induced higher-order EGFR oligomerisation or clustering has been reported but it is not clear how EGFR oligomers, as distinct from EGFR dimers, influence signaling outputs. To address this question, we combined measures of receptor clustering (microscopy; image correlation spectroscopy) and phosphorylation (Western blots) with modelling of mass-action chemical kinetics. A stable BaF/3 cell-line that contains a high proportion (>90%) of inactive dimers of EGFR-eGFP but no secreted ligand and no other detectable ErbB receptors was used as the model cell system. EGF at concentrations of greater than 1 nM was found to cluster EGFR-eGFP dimers into higher-order complexes and cause parallel increases in EGFR phosphorylation. The kinetics of EGFR clustering and phosphorylation were both rapid, plateauing within 2 minutes after stimulation with 30 nM EGF. A rule-based model was formulated to interpret the data. This model took into account ligand binding, ligand-induced conformational changes in the cytosolic tail, monomer-dimer-trimer-tetramer transitions via ectodomain- and kinase-mediated interactions, and phosphorylation. The model predicts that cyclic EGFR tetramers are the predominant phosphorylated species, in which activated receptor dimers adopt a cyclic side-by-side orientation, and that receptor kinase activation is stabilised by the intramolecular interactions responsible for cyclic tetramerization.

Aggregation distributions on cells determined by photobleaching image correlation spectroscopy.

The organization of molecules into macromolecular (nanometer scale), supramolecular complexes (submicron-to-micron scale), and within subcellular domains, is an important architectural principle of cellular biology and biochemistry. Determining the precise nature and distribution of complexes within the cellular milieu is a challenging biophysical problem. Time-series analysis of laser scanning confocal microscopy images by image correlation spectroscopy (ICS) or fluctuation moments methods provides information on aggregation, flow, and dynamics of fluorescently tagged macromolecules. All the methods to date require a brightness standard to relate the experimental data to absolute aggregation. In this article, we show that ICS as a function of gradual photobleaching is a sensitive indicator of aggregation distribution on the submicron scale. Specifically, in photobleaching ICS, the extent of nonlinearity of the apparent cluster density as a function of bleaching is related to the size of clusters. The analysis is tested using computer simulations on model aggregate systems and then applied to an experimental determination of Aß peptide aggregation on nerve cells. The analysis reveals time-dependent increases in Aß1-42 peptide aggregation. Globally, the datasets could be described by a monomer-dimer-tetramer-hexamer or a monomer-dimer-trimer-pentamer model. The results demonstrate the utility of photobleaching with ICS for determining aggregation states on the supramolecular scale in intact cells without the requirement for a brightness standard.