Combination of compound screening with an animal model identifies pentamidine to prevent Chlamydia trachomatis infection.

Chlamydia trachomatis (Ct) is the most common cause for bacterial sexually transmitted infections (STIs) worldwide with a tremendous impact on public health. With the aim to unravel novel targets of the chlamydia life cycle, we screen a compound library and identify 28 agents to significantly reduce Ct growth. The known anti-infective agent pentamidine-one of the top candidates of the screen-shows anti-chlamydia activity in low concentrations by changing the metabolism of host cells impairing chlamydia growth. Furthermore, it effectively decreases the Ct burden upon local or systemic application in mice. Pentamidine also inhibits the growth of Neisseria gonorrhea (Ng), which is a common co-infection of Ct. The conducted compound screen is powerful in exploring antimicrobial compounds against Ct in a medium-throughput format. Following thorough in vitro and in vivo assessments, pentamidine emerges as a promising agent for topical prophylaxis or treatment against Ct and possibly other bacterial STIs.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. METHODS: We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. RESULTS: High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. CONCLUSIONS: Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc.

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

A Bioluminescence-Based Drug Screen Identifies Activities of Fexinidazole and Its Metabolites against Helicobacter pylori.

Helicobacter pylori is responsible for a wide range of gastric diseases, including gastric cancer and gastritis. With half of the world's population infected by H. pylori and the current standard of care associated with suboptimal outcomes, a search for more effective drugs is critical. To facilitate drug screening for H. pylori, we developed a microtiter plate-based compound screening method that is faster and can screen multiple compounds. We identified activities of fexinidazole and its sulfoxide and sulfone metabolites against H. pylori. Both fexinidazole and its metabolites exhibited equipotency against SS1, 60190, and G27 strains, which were about 3-6-fold more potent than the currently used metronidazole. We also determined the minimal inhibitory concentration (MIC) of metronidazole, fexinidazole, and its metabolites against these strains by a traditional agar plate-based method. While MIC values of fexinidazole and metronidazole were similar against all the strains, both sulfoxide and sulfone showed lower MIC values than metronidazole against SS1 and 60190. Given the recent FDA approval of fexinidazole, our data on the in vitro antibacterial activities of fexinidazole and its metabolites support further evaluation of this drug with the goal of producing an alternative nitro-based antimicrobial with good safety profiles for the treatment of H. pylori infection.

BRAF Inhibitor Resistance Confers Increased Sensitivity to Mitotic Inhibitors.

Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies.

Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy.

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

AGMO Inhibitor Reduces 3T3-L1 Adipogenesis.

Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30-100 µM and 5-20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.

Identification of an Antiviral Compound from the Pandemic Response Box that Efficiently Inhibits SARS-CoV-2 Infection In Vitro.

With over 50 million currently confirmed cases worldwide, including more than 1.3 million deaths, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, limited prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, 400 compounds from the antimicrobial "pandemic response box" library were screened for inhibiting properties against SARS-CoV-2. An initial screen on Vero E6 cells identified five compounds that inhibited SARS-CoV-2 replication. However, validation of the selected hits in a human lung cell line highlighted that only a single compound, namely Retro-2.1, efficiently inhibited SARS-CoV-2 replication. Additional analysis revealed that the antiviral activity of Retro-2.1 occurs at a post-entry stage of the viral replication cycle. Combined, these data demonstrate that stringent in vitro screening of preselected compounds in multiple cell lines refines the rapid identification of new potential antiviral candidate drugs targeting SARS-CoV-2.

Epigenetic Compound Screening Uncovers Small Molecules for Reactivation of Latent HIV-1.

During infection with the human immunodeficiency virus type 1 (HIV-1), latent reservoirs are established that circumvent full eradication of the virus by antiretroviral therapy (ART) and are the source for viral rebound after cessation of therapy. As these reservoirs are phenotypically indistinguishable from infected cells, current strategies aim to reactivate these reservoirs, followed by pharmaceutical and immunological destruction of the cells. Here, we employed a simple and convenient cell-based reporter system, which enables sample handling under biosafety level (BSL)-1 conditions, to screen for compounds that were able to reactivate latent HIV-1. The assay showed a high dynamic signal range and reproducibility with an average Z-factor of 0.77, classifying the system as robust. The assay was used for high-throughput screening (HTS) of an epigenetic compound library in combination with titration and cell-toxicity studies and revealed several potential new latency-reversing agents (LRAs). Further validation in well-known latency model systems verified earlier studies and identified two novel compounds with very high reactivation efficiencies and low toxicity. Both drugs, namely, N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) and 2',3'-difluoro-[1,1'-biphenyl]-4-carboxylic acid, 2-butylhydrazide (SR-4370), showed comparable performances to other already known LRAs, did not activate CD4+ T cells, and did not cause changes in the composition of peripheral blood mononuclear cells (PBMCs), as shown by flow cytometry analyses. Both compounds may represent effective new treatment possibilities for reversal of latency in HIV-1-infected individuals.

A gum Arabic assisted sustainable drug delivery system for adult Drosophila.

Large-scale compound screening in adult flies is hampered by the lack of continuous drug delivery systems and poor solubility of numerous compounds. Here we found that gum Arabic (Acacia/Senegal gum), a widely used stabilizer, can also emulsify lipophilic compounds and profoundly increase their accessibility to target tissues in Drosophila and mice. We further developed a gum Arabic-based drug delivery system, wherein the drug was ground into gum Arabic and emulsified in liquid food fed to flies by siphoning through a U-shape glass capillary. This system did not affect food intake nor cell viability. Since drugs were continuously delivered by siphoning, minimal compound waste and less frequent food changes make this system ideal for large-scale long-term screenings. In our pilot screening for antitumor drugs in the NCI DTP library, we used a Drosophila model of colorectal cancer and identified two drugs that are especially hydrophobic and were not identified in previous screenings. Our data demonstrated that gum Arabic facilitates drug delivery in animal models and the system is suitable for long-term high-throughput drug screening in Drosophila This system would accelerate drug discovery for chronic and cognitive conditions.

RNA-seq analysis and compound screening highlight multiple signalling pathways regulating secondary cell death after acute CNS injury in vivo.

Understanding the molecular mechanisms that regulate secondary cell death after acute central nervous system (CNS) injury is critical for the development of effective neuroprotective drugs. Previous research has shown that neurotoxic processes including excitotoxicity, oxidative stress and neuroinflammation can cause secondary cell death. Nevertheless, clinical trials targeting these processes have been largely unsuccessful, suggesting that the signalling pathways underlying secondary cell death remain incompletely understood. Due to their suitability for live imaging and their amenability to genetic and pharmacological manipulation, larval zebrafish provide an ideal platform for studying the regulation of secondary cell death in vivo Here, we use RNA-seq gene expression profiling and compound screening to identify signalling pathways that regulate secondary cell death after acute neural injury in larval zebrafish. RNA-seq analysis of genes upregulated in cephalic mpeg1+ macrophage-lineage cells isolated from mpeg1:GFP transgenic larvae after neural injury suggested an involvement of cytokine and polyamine signalling in secondary cell death. Furthermore, screening a library of FDA approved compounds indicated roles for GABA, serotonin and dopamine signalling. Overall, our results highlight multiple signalling pathways that regulate secondary cell death in vivo, and thus provide a starting point for the development of novel neuroprotective treatments for patients with CNS injury.This article has an associated First Person interview with the two first authors of the paper.

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.

Streamlining drug discovery assays for cardiovascular disease using zebrafish.

Introduction: 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.

Cell-Based Screen Using Amyloid Mimic ß23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of α-Synuclein and Huntingtin Aggregates.

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (ß23) expression model to screen potential lead compounds inhibiting ß23-induced toxicity. Highthroughput screening identified several natural compounds as nuclear ß23 inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic ß23 aggregates and protects SH-SY5Y cells from toxicity induced by ß23 expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and α-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and α-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited α-synuclein aggregation but also disaggregated preformed α-synuclein fibrils in vitro . Taken together, our results suggest that a Tet-Off ß23 cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay.

The capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replication in vitro by decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.

Genome-Scale Signatures of Gene Interaction from Compound Screens Predict Clinical Efficacy of Targeted Cancer Therapies.

Identifying reliable drug response biomarkers is a significant challenge in cancer research. We present computational analysis of resistance (CARE), a computational method focused on targeted therapies, to infer genome-wide transcriptomic signatures of drug efficacy from cell line compound screens. CARE outputs genome-scale scores to measure how the drug target gene interacts with other genes to affect the inhibitor efficacy in the compound screens. Such statistical interactions between drug targets and other genes were not considered in previous studies but are critical in identifying predictive biomarkers. When evaluated using transcriptome data from clinical studies, CARE can predict the therapy outcome better than signatures from other computational methods and genomics experiments. Moreover, the CARE signatures for the PLX4720 BRAF inhibitor are associated with an anti-programmed death 1 clinical response, suggesting a common efficacy signature between a targeted therapy and immunotherapy. When searching for genes related to lapatinib resistance, CARE identified PRKD3 as the top candidate. PRKD3 inhibition, by both small interfering RNA and compounds, significantly sensitized breast cancer cells to lapatinib. Thus, CARE should enable large-scale inference of response biomarkers and drug combinations for targeted therapies using compound screen data.

High-Throughput Functional Genetic and Compound Screens Identify Targets for Senescence Induction in Cancer.

Senescence is a proliferation arrest that can result from a variety of stresses. Cancer cells can also undergo senescence, but the stresses that provoke cancer cells to undergo senescence are unclear. Here, we use both functional genetic and compound screens in cancer cells harboring a reporter that is activated during senescence to find targets that induce senescence. We show that suppression of the SWI/SNF component SMARCB1 induces senescence in melanoma through strong activation of the MAP kinase pathway. From the compound screen, we identified multiple aurora kinase inhibitors as potent inducers of senescence in RAS mutant lung cancer. Senescent melanoma and lung cancer cells acquire sensitivity to the BCL2 family inhibitor ABT263. We propose a one-two punch approach for the treatment of cancer in which a drug is first used to induce senescence in cancer cells and a second drug is then used to kill senescent cancer cells.

Quo natas, Danio?-Recent Progress in Modeling Cancer in Zebrafish.

Over the last decade, zebrafish has proven to be a powerful model in cancer research. Zebrafish form tumors that histologically and genetically resemble human cancers. The live imaging and cost-effective compound screening possible with zebrafish especially complement classic mouse cancer models. Here, we report recent progress in the field, including genetically engineered zebrafish cancer models, xenotransplantation of human cancer cells into zebrafish, promising approaches toward live investigation of the tumor microenvironment, and identification of therapeutic strategies by performing compound screens on zebrafish cancer models. Given the recent advances in genome editing, personalized zebrafish cancer models are now a realistic possibility. In addition, ongoing automation will soon allow high-throughput compound screening using zebrafish cancer models to be part of preclinical precision medicine approaches.

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.