High-throughput Screening Of Small Molecules Research Articles

High-throughput small molecule screening reveals Nrf2-dependent and -independent pathways of cellular stress resistance.

Aging is the dominant risk factor for most chronic diseases. Development of antiaging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in Caenorhabditis elegans, although not in testing performed in worms and flies with a more restricted set of compounds. Transcriptomic analysis and genetic studies implicated Nrf2/SKN-1 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Small molecules identified in this work may represent attractive tools to elucidate mechanisms of stress resistance in mammalian cells.

35 Poster Discussion - High-throughput small molecule screens reveal therapeutic opportunities against TFE3-fusion renal cell carcinoma

35 Poster Discussion - High-throughput small molecule screens reveal therapeutic opportunities against TFE3-fusion renal cell carcinoma

Termi-Luc: a versatile assay to monitor full-protein release from ribosomes.

Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, nonfunctional, or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive approaches are required to study eukaryotic termination mechanisms and to identify potential therapeutics that modulate termination. Canonical radioactivity-based termination assays are complex, report on a short peptide release, and are incompatible with high-throughput screening. Here we describe a robust and simple in vitro assay to study the kinetics of full-protein release. The assay monitors luminescence upon release of nanoluciferase from a mammalian pretermination complex. The assay can be used to record time-progress curves of protein release in a high-throughput format, making it optimal for studying release kinetics and for high-throughput screening for small molecules that modulate the efficiency of termination.

Abstract 6316: High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis

Abstract Lymphangioleiomyomatosis (LAM) is a rare lung cancer characterized by immature smooth muscle-like cell invasion of the lung parenchyma, leading to cystonodular destruction and respiratory decline. Interestingly, LAM is a monogetic disease caused by loss of TSC2, conferring constitutive mTORC1 activation. Currently, there are no approved therapeutics which exhibit LAM cell-specific cytotoxicity. Here, we describe a novel 3D drug screening platform which couples a rationally designed, synthetic, lung-mimetic hydrogel to high content imaging. We have designed and synthesized a hyaluronic acid-based hydrogel which mimics endogenous lung tissue. Human LAM cell models maintain mTORC1 activation in the hydrogel compared to matched controls, reflective of human LAM lesions. We observe LAM cells to exhibit both protease-dependent and independent modes of invasion: the former imparted by gel crosslinking with an MMP-cleavable peptide, the latter enabled by methylcellulose conjugation. Proliferation is inhibited as LAM cells actively invade through the gel. We coupled our culture system to high content confocal microscopy, permitting measurements of cell viability and invasion depth in response to therapeutic screening. We subsequently tested an 800-drug library of Health Canada-approved small molecule cancer therapeutics on LAM cells and matched controls. Surprisingly, LAM cells exhibited pan-therapeutic resistance as measured by both invasion and viability metrics. Enrichment analysis revealed categories of therapeutics which demonstrated LAM-selective cytotoxicity and/or anti-invasiveness. We performed a refinement screen on select therapeutics and identified AURA inhibition as an effective LAM cell-specific therapeutic avenue. Work is ongoing to establish the most efficacious small molecule inhibitor. In conclusion, we describe a novel drug screening platform which enables high content measurements of viability and invasion in response to drug screening in a lung-mimetic system. We have identified AURA inhibition as a potential therapeutic avenue for LAM patients using this screening platform. Future work will involve target validation, elucidation of the mechanism of action, and testing in a pre-clinical mouse model. As these small molecules are Health Canada-approved, we anticipate rapid clinical translation of our preferred candidate. Citation Format: Adam Pietrobon, Julien Yockell-Lelievre, Carole Doré, Roger Y. Tam, Sean P. Delaney, Molly Shoichet, William L. Stanford. High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6316.

Abstract 5043: A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC

Abstract Introduction: Epithelial mesenchymal transition (EMT) and Cancer stem cells (CSCs) are associated with metastasis and tumor recurrence. We previously identified that EMT induces stem cell properties and the transcription factor FOXC2 is one of the key mediators. Recently, in a high throughput small molecule screen using FOXC2-overexpressing cells, we identified FiVe1 that binds to vimentin resulting in mitotic collapse and multinucleation. Vimentin is an intermediate filament that is upregulated during EMT and is present in CSCs, where it provides increased motility as well as resistance to mechanical stress in these cells. In this work, we hypothesize that FiVe1 selectively targets mesenchymal-looking, CSC-enriched populations via vimentin. Experimental procedures: Different subtypes of breast cancer cells and non-transformed cells, as well as CSC-enriched or depleted populations, were assessed for the occurrence of multinucleation (>2 nuclei) after FiVe1 treatment for two population doubling (2PD) through counting by fluorescent microscopy. Cell lines that became multinucleated were then tested for the reversibility of multinucleation after passaging cells with fresh media. Levels of vimentin phosphorylation (S56) and total vimentin were assessed by immunoblot. Summary of data: Multinucleation was observed only in the mesenchymal-like subtypes of TNBC cell lines relative to other TNBC subtypes and non-transformed cell lines. This multinucleation was irreversible upon passaging with fresh media. The levels of S56 phosphorylation of vimentin increased in these cell lines, but gradually decreased upon passaging with fresh media. To determine if multinucleation was due to the higher number of CSCs in mesenchymal-like TNBC cells, CSC-enriched populations were treated with FiVe1. The CSC-enriched populations had significantly increased levels of multinucleation upon FiVe1 treatment relative to CSC-depleted and control populations. Conclusions: The induction of multinucleation by Five1 correlates with higher CSC levels. This was observed both in the mesenchymal-like subtypes of TNBCs, which have higher stem-like properties relative to other TNBC subtypes, as well as cell populations enriched for CSCs. The increase in vimentin phosphorylation at S56 after FiVe1 treatment in TNBC cell lines is physiologically significant as misregulated S56 phosphorylation results in multinucleation and dysregulation of vimentin assembly. While the S56 phosphorylation decreased upon withdrawal of FiVe1 in all cells, the kinetics of reversibility is lower in mesenchymal-looking cells, which corroborate with the multinucleation. Further research regarding the fate of multinucleated cells is in progress. Inhibiting CSCs by targeting vimentin is a promising approach for sensitizing these inherently resistant tumors to chemotherapeutic agents. Citation Format: Nick A. Kuburich, Matthew Rosolen, Francisco Martinez, Petra den Hollander, Michael J. Bollong, Rama Soundararajan, Luke L. Lairson, Sendurai A. Mani. A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5043.

Abstract 457: Discovery and characterization of GNF-7 as a preferentially cytotoxic compound for camptothecin-resistant Ewing sarcoma cells

Abstract Ewing sarcoma (EWS), a highly aggressive bone and soft tissue cancer occurring in children and young adults, is characterized by FET-ETS family gene fusions. Although event-free survival rates remain high in EWS patients with localized disease, those with metastatic or relapsed disease face poor long-term survival odds (under 30%). Our group has been studying the genomic and transcriptomic evolution of drug-resistant EWS cells, specifically when challenged with topoisomerase I inhibitors, a class of chemotherapeutic agents frequently used for relapsed disease. By employing a genome-wide CRISPR knockout library screen, we identified heterozygous deletion of the TOP1 gene, and subsequent decrease of TOP1 protein expression, as the primary method of conferring camptothecin resistance in EW8 cells. To explore vulnerabilities specific to TOP1-deficient cells, we created TOP1-knockdown (TOP1KD) EW8 cells using CRISPR to delete one of the two genomic copies of TOP1. High-throughput small molecule screening was performed on the TOP1KD and parental cell lines to identify compounds with more cytotoxic activity in the TOP1KD cells compared with the TOP1-intact parental cells. The top hit from this screen was GNF-7, a kinase inhibitor originally developed to target multidrug-resistant BCR-ABL in fusion-positive chronic myelogenous leukemia. We verified this hit, showing that GNF-7 had an IC50 that was 10-fold lower in the TOP1KD cells compared to the parental EW8 cells and extended this finding to other EWS cell lines. To explore the mechanism of action of this drug, we treated both parental EW8 and TOP1KD cells with a low dose of GNF-7 for various times and then performed RNAseq. We found that GNF-7 promoted the downregulation of genes induced by the Ewing sarcoma-specific EWS-FLI1 fusion protein in both cell lines. We also subjected GNF-7 to in situ kinase profiling and identified several cellular kinases that were potently inhibited by this compound including CSK, p38α, EphA2, Lyn, and ZAK. We are currently exploring the functional importance of these kinases in the preferential cytotoxicity induced by GNF-7 in TOP1KD cells. Intriguingly, we have demonstrated that pretreatment of EW8 cells with an IC50 dose of camptothecin sensitizes the cells to subsequent GNF-7 treatment. Together, these results suggest that GNF-7 could be an effective treatment for Ewing sarcoma patients in combination with TOP1 inhibitors or for those patients who have developed resistance to TOP1 inhibitors. Citation Format: Carly M. Sayers, Morgan B. Carter, Haiyan Lei, Steven Shema, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jack F. Shern. Discovery and characterization of GNF-7 as a preferentially cytotoxic compound for camptothecin-resistant Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 457.

Abstract 4034: High throughput synergy screening identifies therapeutic combinations for Merkel cell carcinoma

Abstract Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. MCC is virus-positive (VP-MCC) in the majority of cases wherein Merkel cell polyomavirus is integrated into the host genome. The remainder of cases are virus-negative (VN-MCC) and are associated with mutations induced by ultraviolet light. Primary MCC is effectively treated by surgical resection and radiotherapy, but for metastatic MCC, outcomes for conventional chemotherapy are poor. Immune checkpoint inhibitors are more likely to produce durable responses in metastatic MCC, but they are not effective in all cases and are not a viable option for patients with immunosuppression. To address the need for better therapeutic options, we utilized high-throughput small molecule screening to assess the efficacy of ~4,000 drugs in reducing VP-MCC and VN-MCC viability. Among the compounds we screened, navitoclax was identified as an effective agent against VP-MCC cell lines, compared to VN-MCC or control cells. However, navitoclax clinical application is limited by dose-dependent thrombocytopenia. One strategy to reduce thrombocytopenia risk is to combine a lower dose of navitoclax with another MCC-effective agent. To identify effective drug combinations, we performed a synergy screen with 1912 pairings of navitoclax with each of the clinically relevant compounds in the NCATS Mechanism Interrogation PlatE (MIPE). We were able to identify synergy between navitoclax and multiple drug classes. High priority drug combinations are now being evaluated for toxicity and efficacy in pre-clinical xenograft models of MCC. Our study has identified novel therapeutic combinations for the treatment of VP-MCC. By focusing on drugs already in clinical use, validated combinations will be quickly available to patients for whom immunotherapy is not available or effective. Citation Format: Khalid Garman, Tara Gelb, Daniel Urban, Matthew Hall, Isaac Brownell. High throughput synergy screening identifies therapeutic combinations for Merkel cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4034.

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells

BackgroundBreast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR).MethodsWe evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line.ResultsWe found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551.ConclusionsOur data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons

(iScience 23, 100931-1–100931-13; March 27, 2020) In the originally published version of this article, author Ted Kamenecka’s surname was unfortunately misspelled. It appears correctly here and online, and the authors apologize for the error. High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in NeuronsVarkuti et al.iScienceFebruary 21, 2020In BriefChemistry; Biochemistry; Molecular Neuroscience Full-Text PDF Open Access

A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing.

Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.

Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells.

Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

Pharmacological clearance of misfolded rhodopsin for the treatment of RHO-associated retinitis pigmentosa.

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening (HTS) of 68,979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of RhoP23H/+ knock-in mice at 1month of age. Additionally, four weekly IVIs increased the photoreceptor cell number in the retinae of RhoP23H/+ mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin-associated RP.

Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release

BackgroundThe continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production.ResultsThe reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester.ConclusionThese results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most γ-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Inhibition of BCL2 Family Members Increases the Efficacy of Copper Chelation in BRAFV600E-Driven Melanoma.

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. SIGNIFICANCE: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.

Screening for YAP Inhibitors Identifies Statins as Modulators of Fibrosis.

Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.

Exploring the Binding Potency and Specificity of Small Molecules Against the Transmembrane Amyloid Precursor Protein Fragment, C99

The Amyloid Precursor Protein (APP) is a ubiquitously expressed single-pass transmembrane receptor that is involved in a variety of roles ranging from neurological development to synaptic plasticity and homeostasis. Despite several biological functions, APP is most famous for being the precursor of Amyloid-β (Aβ) peptides, a hypothesized cause of Alzheimer's disease (AD; Amyloid Cascade Hypothesis). 30 years of research on APP have passed yet there are no reported chemical probes that can bind APP in a potent and specific way. The 3D structure of the βAPP-CTF (C99; the C-terminal 99-residue protein product of APP cleavage by β-secretase) revealed a hydrophobic, transmembrane pseudo-pocket that binds cholesterol, motivating the concept that C99 may be able to bind other hydrophobic small-molecules. Here I describe the results of NMR-based high-throughput screening (HTS) of small-molecules for compounds that bind avidly and specifically to C99 in model membranes.

A High-Throughput Small Molecule Screen Identifies Ouabain as Synergistic with miR-34a in Killing Lung Cancer Cells.

SummaryMicroRNA-34 (miR-34) is one of the major families of tumor suppressor miRNAs often lost in cancers. Delivery of miR-34a mimics to affected tumors as a therapeutic strategy has been tried in pre-clinical studies and in a phase I clinical trial. One approach to increase efficacy and reduce toxicity is to rationally identify drug combinations with small molecules that synergize with miR-34a. In this study we performed a high-throughput screen of a large panel of small molecules with known biological activity and identified ouabain as a candidate small molecule that synergized with miR-34a in killing lung cancer cells. We elucidated autophagy activation as a key mechanism by which miR-34a and ouabain causes increased cytotoxicity in cells. We posit that this combinatorial approach could reduce the active dose of miR-34a needed in vivo to observe tumor shrinkage and potentiate the development of miR-34a combination therapies in the future.

P145 MECHANISMS OF ACTION FOR THE IBD-RISK GENE C1ORF106/INAVA

Abstract C1ORF106, also named INAVA (Innate Immune Activator), was identified as a risk gene for the chronic inflammatory bowel diseases (IBD) by genome-wide association studies and targeted exome sequencing. INAVA is highly enriched in polarized simple epithelia, the cell type that lines the intestine and interfaces most directly with the outside world. We discover that INAVA provides a direct mechanistic link between mucosal barriers and inflammation. This is driven by INAVA’s signature Domain of Unknown Function DUF3338, which we newly defined as an enhancer of IL-1β-TRAF6 polyubiquitination cascades. Our work shows that DUF3338 stably binds the GTP-exchange factor cytohesin-2 (ARNO), in one case blocking the formation of INAVA stress puncta and its effect on ubiquitination, and in another case acting at lateral membranes where the INAVA-ARNO complex regulates cortical F-actin dynamics and epithelial barrier function. We thus rename the DUF3338 domain CUPID for Cytohesin Ubiquitin Protein Inducing Domain. We performed a high throughput small molecule screen to identify molecules that modulated INAVA function in cells. Compounds hits identified in the screen links INAVA’s role in barrier and stress response mechanisms involving actin dynamics and proteostasis.

Drug Screen Tugs at Common Thread for Repeat Disorders

Repeat-associated non-ATG (RAN) translation is emerging as a driver of pathogenesis in microsatellite expansion disorders. Green and colleagues recently identified several candidate RAN translation inhibitors from a high-throughput small-molecule screen for fragile X tremor ataxia syndrome. Their study establishes a path forward for identifying inhibitors of RAN translation for multiple disorders.