High-throughput Chemical Screening Research Articles

CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing.

SF3B1 is the most frequently mutated splicing factor in cancer. Mutations in SF3B1 likely confer clonal advantages to cancer cells but they may also confer vulnerabilities that can be therapeutically targeted. SF3B1 cancer mutations can be maintained in homozygosis in C. elegans, allowing synthetic lethal screens with a homogeneous population of animals. These mutations cause alternative splicing (AS) defects in C. elegans, as it occurs in SF3B1-mutated human cells. In a screen, we identified RNAi of U2 snRNP components that cause synthetic lethality with sftb-1/SF3B1 mutations. We also detected synthetic interactions between sftb-1 mutants and cancer-related mutations in uaf-2/U2AF1 or rsp-4/SRSF2, demonstrating that this model can identify interactions between mutations that are mutually exclusive in human tumors. Finally, we have edited an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Thus, we have established a multicellular model for SF3B1 mutations amenable for high-throughput genetic and chemical screens.

Streamlining drug discovery assays for cardiovascular disease using zebrafish

ABSTRACTIntroduction: In the last decade, our armamentarium of cardiovascular drug therapy has expanded significantly. Using innovative functional genomics strategies such as genome editing by CRISPR/Cas9 as well as high-throughput assays to identify bioactive small chemical compounds has significantly facilitated elaboration of the underlying pathomechanism in various cardiovascular diseases. However, despite scientific progress approvals for cardiovascular drugs has stagnated significantly compared to other fields of drug discovery and therapy during the past years.Areas covered: In this review, the authors discuss the aspects and pitfalls during the early phase of cardiovascular drug discovery and describe the advantages of zebrafish as an in vivo organism to model human cardiovascular diseases (CVD) as well as an in vivo platform for high-throughput chemical compound screening. They also highlight the emerging, promising techniques of automated read-out systems during high-throughput screening (HTS) for the evaluation of important cardiac functional parameters in zebrafish with the potential to streamline CVD drug discovery.Expert opinion: The successful identification of novel drugs to treat CVD is a major challenge in modern biomedical and clinical research. In this context, the definition of the etiologic fundamentals of human cardiovascular diseases is the prerequisite for an efficient and straightforward drug discovery.

High-throughput Chemical Screening Identifies Focal Adhesion Kinase and Aurora Kinase B Inhibition as a Synergistic Treatment Combination in Ewing Sarcoma.

Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients. Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma. Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma. FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Matrix Linear Models for High-Throughput Chemical Genetic Screens.

We develop a flexible and computationally efficient approach for analyzing high-throughput chemical genetic screens. In such screens, a library of genetic mutants is phenotyped in a large number of stresses. Typically, interactions between genes and stresses are detected by grouping the mutants and stresses into categories, and performing modified t-tests for each combination. This approach does not have a natural extension if mutants or stresses have quantitative or nonoverlapping annotations (e.g., if conditions have doses or a mutant falls into more than one category simultaneously). We develop a matrix linear model (MLM) framework that allows us to model relationships between mutants and conditions in a simple, yet flexible, multivariate framework. It encodes both categorical and continuous relationships to enhance detection of associations. We develop a fast estimation algorithm that takes advantage of the structure of MLMs. We evaluate our method's performance in simulations and in an Escherichia coli chemical genetic screen, comparing it with an existing univariate approach based on modified t-tests. We show that MLMs perform slightly better than the univariate approach when mutants and conditions are classified in nonoverlapping categories, and substantially better when conditions can be ordered in dosage categories. Therefore, it is an attractive alternative to current methods, and provides a computationally scalable framework for larger and complex chemical genetic screens. A Julia language implementation of MLMs and the code used for this paper are available at https://github.com/janewliang/GeneticScreen.jl and https://bitbucket.org/jwliang/mlm_gs_supplement, respectively.

Combination of PARP inhibitor and temozolomide to suppress chordoma progression

Chordoma, a malignant bone cancer, is highly resistant to conventional therapeutic approaches; this greatly limits radio- and chemotherapeutic options and disease management. In the present study, we investigated three patient-derived chordoma cell lines to elucidate the molecular mechanism of resistance to therapeutics. An in vitro high-throughput chemical screening assay and an in vivo xenograft model were used to identify novel chemosensitizers for chordoma. We found that patient-derived chordoma cell lines recapitulated disease phenotypes, which were highlighted by robust resistance to medical therapy manifested as lack of DNA damage accumulation. Mechanistically, the PARP DNA repair pathway was found to play a central role in this resistance. Chemical screening confirmed that PARP inhibitors could strikingly enhance temozolomide (TMZ) therapy in chordoma cells. Combining the FDA-approved PARP inhibitor, olaparib, with chemotherapeutics not only potentiated DNA damage accumulation, cell cycle arrest, and apoptosis in vitro but also suppressed chordoma xenograft expansion in vivo. We conclude that combining PARP inhibition with TMZ could be an effective therapeutic approach for the clinical management of chordoma. KEY MESSAGES: The PARP DNA repair pathway enhances chemoresistance in chordoma cells. Combining PARP inhibitors with genotoxic agents induces chordoma cell cytotoxicity. PARP inhibitor combining with temozolomide suppresses growth of chordoma in vivo.

Chemical screen for epigenetic barriers to single allele activation of Oct4

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.

Targeting Super-Enhancer-Driven Oncogenic Transcription by CDK7 Inhibition in Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive malignancies, with no effective treatment currently available. The molecular mechanisms of ATC carcinogenesis remain poorly understood. The objective of this study was to investigate the mechanisms and functions of super-enhancer (SE)-driven oncogenic transcriptional addiction in the progression of ATC and identify new drug targets for ATC treatments. Methods: High-throughput chemical screening was performed to identify new drugs inhibiting ATC cell growth. Cell viability assay, colony formation analysis, cell-cycle analysis, and animal study were used to examine the effects of drug treatments on ATC progression. Chromatin immunoprecipitation sequencing was conducted to establish a SE landscape of ATC. Integrative analysis of RNA sequencing, chromatin immunoprecipitation sequencing, and CRISPR/Cas9-mediated gene editing was used to identify THZ1 target genes. Drug combination analysis was performed to assess drug synergy. Patient samples were analyzed to evaluate candidate biomarkers of prognosis in ATC. Results: THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), was identified as a potent anti-ATC compound by high-throughput chemical screening. ATC cells, but not papillary thyroid carcinoma cells, are exceptionally sensitive to CDK7 inhibition. An integrative analysis of both gene expression profiles and SE features revealed that the SE-mediated oncogenic transcriptional amplification mediates the vulnerability of ATC cells to THZ1 treatment. Combining this integrative analysis with functional assays led to the discovery of a number of novel cancer genes of ATC, including PPP1R15A, SMG9, and KLF2. Inhibition of PPP1R15A with Guanabenz or Sephin1 greatly suppresses ATC growth. Significantly, the expression level of PPP1R15A is correlated with CDK7 expression in ATC tissue samples. Elevated expression of PPP1R15A and CDK7 are both associated with poor clinical prognosis in ATC patients. Importantly, CDK7 or PPP1R15A inhibition sensitizes ATC cells to conventional chemotherapy. Conclusions: Taken together, these findings demonstrate transcriptional addiction in ATC pathobiology and identify CDK7 and PPP1R15A as potential biomarkers and therapeutic targets for ATC.

Leukemia Cell Cycle Chemical Profiling Identifies the G2-Phase Leukemia Specific Inhibitor Leusin-1.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow-cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181420 druglike compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2 phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.

Live-imaging Analyses Using Small Fish Models Reveal New Mechanisms That Regulate Primary Tumorigenesis

Since the 1980s, zebrafish (Danio rerio) have been used as a valuable model system to investigate developmental processes because they: 1) grow outside their mothers; 2) are transparent during the embryonic stage; and 3) have organs similar to those in humans. Recently, zebrafish have emerged as a powerful model animal for studying not only developmental biology but also human diseases, especially cancer. Owing to the significant advantages of zebrafish, such as low-cost breeding, high efficiency of transgenesis, and ease of in vivo imaging and oncogenic/tumor cell induction, zebrafish offer a unique opportunity to unveil novel mechanisms of cancer progression, invasion, and metastasis. In addition, the small size of zebrafish larvae enables high-throughput chemical screening, and this advantage contributes to generating useful platforms for antitumor drug discovery. Owing to these various merits, which other model animals (such as fly, mouse, and rat) do not possess, zebrafish could achieve a unique status in cancer research. In this review, we discuss the availability of zebrafish for studying cancer and introduce recent cancer studies that have used zebrafish.

High-throughput screening and classification of chemicals and their effects on neuronal gene expression using RASL-seq

We previously used RNA-seq to identify chemicals whose effects on neuronal gene expression mimicked transcriptional signatures of autism, aging, and neurodegeneration. However, this approach was costly and time consuming, which limited our study to testing a single chemical concentration on mixed sex cortical neuron cultures. Here, we adapted a targeted transcriptomic method (RASL-seq, similar to TempO-seq) to interrogate changes in expression of a set of 56 signature genes in response to a library of 350 chemicals and chemical mixtures at four concentrations in male and female mouse neuronal cultures. This enabled us to replicate and expand our previous classifications, and show that transcriptional responses were largely equivalent between sexes. Overall, we found that RASL-seq can be used to accelerate the pace at which chemicals and mixtures that transcriptionally mimic autism and other neuropsychiatric diseases can be identified, and provides a cost-effective way to quantify gene expression with a panel of marker genes.

High-throughput screening and classification of chemicals and their effects on neuronal gene expression using RASL-seq

High-throughput screening and classification of chemicals and their effects on neuronal gene expression using RASL-seq

High Throughput Chemical Screening Reveals Multiple Regulatory Proteins on FOXA1 in Breast Cancer Cell Lines.

Forkhead box A1 (FOXA1) belongs to the forkhead class transcription factor family, playing pioneering function for hormone receptors in breast and prostate cancers, and mediating activation of linage specific enhancers. Interplay between FOXA1 and breast cancer specific signaling pathways has been reported previously, indicating a regulation network on FOXA1 in breast cancer cells. Here in this study, we aimed to identify which are the proteins that could potentially control FOXA1 function in breast cancer cell lines expressing different molecular markers. We first established a luciferase reporter system reflecting FOXA1 binding to DNA. Then, we applied high throughput chemical screening of multiple protein targets and mass spectrometry in breast cancer cell lines expressing different molecular markers: ER positive/HER2 negative (MCF-7), ER positive/HER2 positive (BT474), and ER negative/HER2 positive (MDA-MB-453). Regardless of estrogen receptor status, HER2 (human epidermal growth factor receptor 2) enriched cell lines showed similar response to kinase inhibitors, indicating the control of FOXA1 by cell signaling kinases. Among these kinases, we identified additional receptor tyrosine kinases and cyclin-dependent kinases as regulators of FOXA1. Furthermore, we performed proteomics experiments from FOXA1 inmunoprecipitated protein complex to identify that FOXA1 interacts with several proteins. Among all the targets, we identified cyclin-dependent kinase 1 (CDK1) as a positive factor to interact with FOXA1 in BT474 cell line. In silico analyses confirmed that cyclin-dependent kinases might be the kinases responsible for FOXA1 phosphorylation at the Forkhead domain and the transactivation domain. These results reveal that FOXA1 is potentially regulated by multiple kinases. The cell cycle control kinase CDK1 might control directly FOXA1 by phosphorylation and other kinases indirectly by means of regulating other proteins.

The Utility of Zebrafish as a Model for Screening Developmental Neurotoxicity.

The developing central nervous system and the blood brain barrier are especially vulnerable and sensitive to different chemicals, including environmental contaminants and drugs. Developmental exposure to these compounds has been involved in several neurological disorders, such as autism spectrum disorders as well as Alzheimer’s and Parkinson’s diseases. Zebrafish (Danio Rerio) have emerged as powerful toxicological model systems that can speed up chemical hazard assessment and can be used to extrapolate neurotoxic effects that chemicals have on humans. Zebrafish embryos and larvae are convenient for high-throughput screening of chemicals, due to their small size, low-cost, easy husbandry, and transparency. Additionally, zebrafish are homologous to other higher order vertebrates in terms of molecular signaling processes, genetic compositions, and tissue/organ structures as well as neurodevelopment. This mini review underlines the potential of the zebrafish as complementary models for developmental neurotoxicity screening of chemicals and describes the different endpoints utilized for such screening with some studies illustrating their use.

High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

Identification of Novel Pathways and Small Molecules Able to Down Regulate Oncogenes Expression By Compounds Screening Approaches in Acute Leukemia and Neuroblastoma Cells

Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, resistance to ALK inhibitors is a ubiquitous problem in ALK-expressing cell lines as well as treated patients. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. Previous reports have suggested that mechanism of resistance is mediated by mutations in the ALK kinase domain impairing binding of an inhibitor to an ALK protein. Thus, new treatment modalities are urgently needed to sensitize patients to crizotinib thereby improving the management of hematological or solid malignancies harboring ALK mutations. To identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, we implemented a high throughput chemical screen in 4 NB-derived cell lines, using a curated library of ~450 compounds. In the compounds screening, JAK-STAT kinase inhibitor (cucurbitacin I) was the most discriminatory with regard to sensitivity for ALK-mutated cell lines. Since Gamma cytokine JAK/STAT system as a target in the treatment of T-cell or myeloid malignancies, we analyzed the cytotoxicity of cucurbitacin I antitumor effect using an MTT assay revealed cucurbitacin I to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by cucurbitacin I (IC50values:T-ALL lines, ICH-TALL-UK, 103.2 nM; MOLT-14, 35.6 nM; KCMC-T, 134.2 nM; Jurkat, 77 nM; ICH-TALL-SM, 47.7 nM; MOLT-4, 22.2 nM. AML lines, HEL, 66.7 nM; Kasumi-3, 4.5 nM; KG-1, 53.6 nM; THP-1, 337.2 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of cucurbitacin I. In NB cell lines harboring F1174L or R1275Q-mutated ALK, crizotinib combined with cucurbitacin I enhanced tumor responses and showed synergistic cytotoxicity.Although crizotinib and cucurbitacin I alone or combination therapy (cucurbitacin I + crizotinib) did not result in decreased viability over control compared with vehicle, the combination therapy in all of 6 cell lines with ALK aberrations and 10 of 13 ALK wild-type cell lines with MYCN amplification or 11q LOH was more effective than vehicle, crizotinib alone, and cucurbitacin I alone. Analysis of downstream signalling through MAPK, AKT and STAT3 pathways showed that NIH3T3 cells stably expressed F1174L mutated ALK or TGW cells harboring R1275Q-mutated ALK, expressed lower levels of pERK, pAKT and pSTAT3 in combination therapy compared with cells treated with cucurbitacin I or crizotinib alone. These findings may provide a indication that the combination of low-dose ALK and STAT3 inhibitors may be benefitical for the treatment of NB, by enhancing efficacy while reducing toxicity. In conclusion, our studies suggested that NB, T-ALL or AML cell lines also exhibited potent cytotoxic responses to the cucurbitacin I and the combination of ALK and JAK-STAT inhibitors could be a valuable therapeutic option for ALK mutated malignancies such as high-risk NB with potential clinical application. DisclosuresNo relevant conflicts of interest to declare.

A fluorescent 3D cell culture assay for high throughput screening of cancer drugs down-regulating survivin

Survivin, a member of inhibitor of apoptosis family, is currently undergoing intensive investigations as a promising cancer marker due to its overexpression in multiple tumor tissues and close relationship with chemotherapy resistance. In this study, a novel 3D survivin promoter assay was developed, using enhanced green fluorescent protein (EGFP) as the reporter to assess survivin promoter activity for cancer drug screening. Breast cancer MCF-7 cells were engineered to express EGFP controlled by a human survivin promoter and a CMV promoter, respectively. These cells were cultured in three-dimensional (3D) polymer-based scaffolds on a 40-microbioreactor platform (40-MBR) with real-time monitoring of EGFP signals. The EGFP production driven by the survivin promoter was strongly correlated with survivin transcriptional level in MCF-7 cells treated with YM155, a small-molecule survivin promoter suppressant. Moreover, the potential inhibition effects of doxorubicin and cisplatin on survivin and their cytotoxicity were also evaluated in this system. This study demonstrated the potential application of the novel 3D survivin promoter-EGFP reporter assay for high-throughput screening of chemicals down-regulating survivin as a molecular target for cancer therapy.

Time-dependent behavioral data from zebrafish reveals novel signatures of chemical toxicity using point of departure analysis

High-content imaging of larval zebrafish behavior can be used as a screening approach to rapidly evaluate the relative potential for chemicals to cause toxicity. However, most statistical methods applied to these data transform movement values to incidence-based “hits” and calculate lowest effect levels (LELs), which loses individual fish resolution of behavior and defies hazard ranking due to reliance on applied dose levels. We developed a parallelizable workflow to calculate benchmark dose (BMD) values from dynamic, high-content zebrafish behavior data that scales for high-throughput chemical screening. To capture the zebrafish movement response from light to dark stimulus, we summarized time-dependent data using both area under the curve and the immediate change at the transition point into two novel metrics that characterized abnormal behavior as a function of chemical concentration. The BMD workflow was applied to calculate BMD10 values of 1060 ToxCast chemicals for 24 zebrafish endpoints, including behavior, mortality and morphology. The BMD10 values provided better precision and separation than LELs for clustering chemicals since they were derived from models that best-fit their concentration-response curves. Analysis of BMD10 values revealed behavioral signatures as the most sensitive endpoints. High concordance in chemical activity was observed between ToxCast in vitro data and zebrafish in vivo behavioral data, however ToxPi analysis indicated that rankings based on in vitro data were not a reliable predictor of in vivo rankings for lower potency chemicals. This analysis method will enable the use of high-content zebrafish behavioral screening data for BMD analysis in toxicological hazard assessment.

Drosophila melanogaster as a function-based high-throughput screening model for antinephrolithiasis agents in kidney stone patients

ABSTRACTKidney stone disease involves the aggregation of stone-forming salts consequent to solute supersaturation in urine. The development of novel therapeutic agents for this predominantly metabolic and biochemical disorder have been hampered by the lack of a practical pre-clinical model amenable to drug screening. Here, Drosophila melanogaster, an emerging model for kidney stone disease research, was adapted as a high-throughput functional drug screening platform independent of the multifactorial nature of mammalian nephrolithiasis. Through functional screening, the therapeutic potential of a novel compound commonly known as arbutin that specifically binds to oxalate, a key component of kidney calculi, was identified. Through isothermal titration calorimetry, high-performance liquid chromatography and atomic force microscopy, arbutin was determined to interact with calcium and oxalate in both free and bound states, disrupting crystal lattice structure, growth and crystallization. When used to treat patient urine samples, arbutin significantly abrogated calculus formation in vivo and outperformed potassium citrate in low pH urine conditions, owing to its oxalate-centric mode of action. The discovery of this novel antilithogenic compound via D. melanogaster, independent of a mammalian model, brings greater recognition to this platform, for which metabolic features are primary outcomes, underscoring the power of D. melanogaster as a high-throughput drug screening platform in similar disorders. This is the first description of the use of D. melanogaster as the model system for a high-throughput chemical library screen. This article has an associated First Person interview with the first authors of the paper.

Dynamin impacts homology-directed repair and breast cancer response to chemotherapy.

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.

Incorporating Lifecycle Emission Information in Promoting Chemical Exposure Screening

While high-throughput human chemical exposure screening often relies on hazard indicators such as persistence and bioaccumulation potential, realistic human chemical exposure and potential health risks are also dependent on factors such as chemical use information and chemical-specific exposure pathways. Multimedia, multi-pathway exposure models are being used for chemical prioritization and screening, the parameterization of which requires chemical specific use and emission information (e.g., how is it emitted? how much?). This information, however, is often insufficient or missing for most chemicals in commerce. To address this issue, we propose integrating substance flow analysis, which is a powerful tool to derive chemical emissions throughout the chemical/product lifecycle, into high-throughput screening for human exposure potential. As an illustrative example, we combine a substance flow model (a simplified version of CiP-CAFE) with chemical fate and exposure models (RAIDAR and RAIDAR-ICE), to create a modeling continuum from the quantity produced or imported to human aggregate exposure. Our model requires inputs of basic properties of chemicals (e.g., partitioning coefficients, and degradation/biotransformation rates) and associated products (e.g., use categories, lifespans and material characteristics). These properties can either be retrieved from existing data resources or calculated by models, which enables the model's wide applicability to high-throughput screening of both existing and new substances. The model allows "forward" evaluation of human aggregate exposure to chemicals arising from their actual use, or "back" calculation of the critical production/import quantities of a chemical that would not cause an unacceptable level of risk (the latter depends on the availability of toxicological data). Overall, the approach demonstrates promise in high-throughput exposure screening of the myriad of chemicals.