Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Optimization of Cell Viability Assays for Drug Sensitivity Screens.

During the preclinical stages of the drug discovery process, cell viability assays are fundamental tools for studying the phenotypic properties and overall health of cells following in vitro drug sensitivity screens. Therefore, it is important to optimize your viability assay of choice to obtain reproducible and replicable results, as well as use relevant drug response metrics (e.g., IC50, AUC, GR50, and GRmax) to identify candidate drugs for further evaluation in vivo. Herein, we used the resazurin reduction assay which is a quick, cost-effective, simple-to-use, and sensitive method for examining the phenotypic properties of cells. Using the MCF7 breast cancer cell line, we provide a detailed step-by-step protocol for optimizing drug sensitivity screens using the resazurin assay.

Luminescence Energy Transfer-Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery.

Luminescence is characterized by the spontaneous emission of light resulting from either chemical or biological reactions. Because of their high sensitivity, reduced background interference, and applicability to numerous situations, luminescence-based assay strategies play an essential role in early-stage drug discovery. Newer developments in luminescence-based technologies have dramatically affected the ability of researchers to investigate molecular binding events. At the forefront of these developments are the nano bioluminescence resonance energy transfer (NanoBRET) and amplified luminescent proximity homogeneous assay (Alpha) technologies. These technologies have opened up numerous possibilities for analyzing the molecular biophysical properties of complexes in environments such as cell lysates. Moreover, NanoBRET enables the validation and quantitation of the interactions between therapeutic targets and small molecules in live cells, representing an essential benchmark for preclinical drug discovery. Both techniques involve proximity-based luminescence energy transfer, in which excited-state energy is transferred from a donor to an acceptor, where the efficiency of transfer depends on proximity. Both approaches can be applied to high-throughput compound screening in biological samples, with the NanoBRET assay providing opportunities for live-cell screening. Representative applications of both technologies for assessing physical interactions and associated challenges are discussed.

Identification of Beilschmiedia tsangii Root Extract as a Liver Cancer Cell-Normal Keratinocyte Dual-Selective NRF2 Regulator.

Transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) plays a crucial role in regulating the expression of genes participating in cellular defense mechanisms against oxidative or xenobiotic insults. However, there is increasing evidence showing that hyperactivation of NRF2 is associated with chemoresistance in several cancers, including hepatocellular carcinoma (HCC), thus making NRF2 an attractive target for cancer therapy. Another important issue in cancer medication is the adverse effects of these substances on normal cells. Here, we attempted to identify a dual-selective NRF2 regulator that exerts opposite effects on NRF2-hyperactivated HCC cells and normal keratinocytes. An antioxidant response element driven luciferase reporter assay was established in Huh7 and HaCaT cells as high-throughput screening platforms. Screening of 3,000 crude extracts from the Taiwanese Indigenous Plant Extract Library resulted in the identification of Beilschmiedia tsangii (BT) root extract as a dual-selective NRF2 regulator. Multiple compounds were found to contribute to the dual-selective effects of BT extract on NRF2 signaling in two cell lines. BT extract reduced NRF2 protein level and target gene expression levels in Huh7 cells but increased them in HaCaT cells. Furthermore, notable combinatory cytotoxic effects of BT extract and sorafenib on Huh7 cells were observed. On the contrary, sorafenib-induced inflammatory reactions in HaCaT cells were reduced by BT extract. In conclusion, our results suggest that the combination of a selective NRF2 activator and inhibitor could be a practical strategy for fine-tuning NRF2 activity for better cancer treatment and that plant extracts or partially purified fractions could be a promising source for the discovery of dual-selective NRF2 regulators.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays.

Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

Drug-nutrient interactions: discovering prescription drug inhibitors of the thiamine transporter ThTR-2 (SLC19A3).

BACKGROUND: Transporter-mediated drug-nutrient interactions have the potential to cause serious adverse events. However, unlike drug-drug interactions, these drug-nutrient interactions receive little attention during drug development. The clinical importance of drug-nutrient interactions was highlighted when a phase III clinical trial was terminated due to severe adverse events resulting from potent inhibition of thiamine transporter 2 (ThTR-2; SLC19A3). OBJECTIVE: In this study, we tested the hypothesis that therapeutic drugs inhibit the intestinal thiamine transporter ThTR-2, which may lead to thiamine deficiency. METHODS: For this exploration, we took a multifaceted approach, starting with a high-throughput in vitro primary screen to identify inhibitors, building in silico models to characterize inhibitors, and leveraging real-world data from electronic health records to begin to understand the clinical relevance of these inhibitors. RESULTS: Our high-throughput screen of 1360 compounds, including many clinically used drugs, identified 146 potential inhibitors at 200 µM. Inhibition kinetics were determined for 28 drugs with half-maximal inhibitory concentration (IC50) values ranging from 1.03 µM to >1 mM. Several oral drugs, including metformin, were predicted to have intestinal concentrations that may result in ThTR-2-mediated drug-nutrient interactions. Complementary analysis using electronic health records suggested that thiamine laboratory values are reduced in individuals receiving prescription drugs found to significantly inhibit ThTR-2, particularly in vulnerable populations (e.g., individuals with alcoholism). CONCLUSIONS: Our comprehensive analysis of prescription drugs suggests that several marketed drugs inhibit ThTR-2, which may contribute to thiamine deficiency, especially in at-risk populations.

Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

The in vivo antinociceptive and µ-opioid receptor activating effects of the combination of N-phenyl-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amines and naloxone.

Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of µ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem, we sought alternative ways to activate MOR - either by use of novel ligands, or via a novel activation mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15 cell-based FLIPR® calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100 mg/kg of compound 5j elicited antinociception in a mouse tail-flick model with an ED50 of 17.5 ±â€¯4 mg/kg. These results strongly suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study, as its discovery has the potential to yield an entirely novel class of analgesics.

Statistical assessment and visualization of synergies for large-scale sparse drug combination datasets.

BACKGROUND: Drug combinations have the potential to improve efficacy while limiting toxicity. To robustly identify synergistic combinations, high-throughput screens using full dose-response surface are desirable but require an impractical number of data points. Screening of a sparse number of doses per drug allows to screen large numbers of drug pairs, but complicates statistical assessment of synergy. Furthermore, since the number of pairwise combinations grows with the square of the number of drugs, exploration of large screens necessitates advanced visualization tools. RESULTS: We describe a statistical and visualization framework for the analysis of large-scale drug combination screens. We developed an approach suitable for datasets with large number of drugs pairs even if small number of data points are available per drug pair. We demonstrate our approach using a systematic screen of all possible pairs among 108 cancer drugs applied to melanoma cell lines. In this dataset only two dose-response data points per drug pair and two data points per single drug test were available. We used a Bliss-based linear model, effectively borrowing data from the drug pairs to obtain robust estimations of the singlet viabilities, consequently yielding better estimates of drug synergy. Our method improves data consistency across dosing thus likely reducing the number of false positives. The approach allows to compute p values accounting for standard errors of the modeled singlets and combination viabilities. We further develop a synergy specificity score that distinguishes specific synergies from those arising with promiscuous drugs. Finally, we developed a summarized interactive visualization in a web application, providing efficient access to any of the 439,000 data points in the combination matrix ( http://www.cmtlab.org:3000/combo_app.html ). The code of the analysis and the web application is available at https://github.com/arnaudmgh/synergy-screen . CONCLUSIONS: We show that statistical modeling of single drug response from drug combination data can help determine significance of synergy and antagonism in drug combination screens with few data point per drug pair. We provide a web application for the rapid exploration of large combinatorial drug screen. All codes are available to the community, as a resource for further analysis of published data and for analysis of other drug screens.

A medium-throughput screen for inhibitors of human metapneumovirus.

Human metapneumovirus, a paramyxovirus discovered in 2001, is a major cause of lower respiratory infection in adults and children worldwide. There are no licensed vaccines or drugs for human metapneumovirus. We developed a fluorescent, cell-based medium-throughput screening assay for human metapneumovirus that captures inhibitors of all stages of the viral lifecycle except budding of progeny virus particles from the cell membrane. We optimized and validated the assay and performed a successful medium-throughput screening. A number of hits were identified, several of which were confirmed to inhibit viral replication in secondary assays. This assay offers potential to discover new antivirals for human metapneumovirus and related respiratory viruses. Compounds discovered using the medium-throughput screening may also provide useful probes of viral biology.

Screening-Based Chemical Approaches to Unravel Stem Cell Biology.

Cell-permeable compounds provide a convenient and efficient approach to manipulate biological processes. A number of compounds controlling stem cell self-renewal, survival, differentiation, and reprogramming have been identified through high-throughput/content screens. Using these powerful chemical tools, strategies have been developed to direct human pluripotent stem cell (hPSC) differentiation to functional cells. Recently, hPSC-derived cells and organoids are used to model human diseases, which can be adapted to a high-throughput/content platform for chemical screens. The identified compounds provide novel tools for decoding the signaling pathways regulating disease progression and candidates for facilitating future drug discovery. Moreover, humanized mouse models carrying hPSC-derived cells enable an innovative system to evaluate the long-term in vivo efficacy of drug candidates on human cells. In summary, screening-based chemical approaches not only expedite strategy development of controlling stem cell fates, but also provide powerful tools for dissecting the molecular mechanisms regulating disease progression.

A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants.

Drug repurposing offers an expedited and economical route to develop new clinical therapeutics in comparison to traditional drug development. Growth-based high-throughput screening is concomitant with drug repurposing and enables rapid identification of new therapeutic uses for investigated drugs; however, this traditional method is not compatible with microorganisms with abnormal growth patterns such as Staphylococcus aureus small-colony variants (SCV). SCV subpopulations are auxotrophic for key compounds in biosynthetic pathways, which result in low growth rate. SCV formation is also associated with reduced antibiotic susceptibility, and the SCV's ability to revert to the normal cell growth state is thought to contribute to recurrence of S. aureus infections. Thus, there is a critical need to identify antimicrobial agents that are potent against SCV in order to effectively treat chronic infections. Accordingly, here we describe adapting an adenylate kinase (AK)-based cell death reporter assay to identify members of a Food and Drug Administration (FDA)-approved drug library that display bactericidal activity against S. aureus SCV. Four library members, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, exhibited potent SCV bactericidal activity against a stable S. aureus SCV. Further investigation showed that sitafloxacin was potent against methicillin-susceptible and -resistant S. aureus, as well as S. aureus within an established biofilm. Taken together, these results demonstrate the ability to use the AK assay to screen small-molecule libraries for SCV bactericidal agents and highlight the therapeutic potential of sitafloxacin to be repurposed to treat chronic S. aureus infections associated with SCV and/or biofilm growth states.IMPORTANCE Conventional antibiotics fail to successfully treat chronic osteomyelitis, endocarditis, and device-related and airway infections. These recurring infections are associated with the emergence of SCV, which are recalcitrant to conventional antibiotics. Studies have investigated antibiotic therapies to treat SCV-related infections but have had little success, emphasizing the need to identify novel antimicrobial drugs. However, drug discovery is a costly and time-consuming process. An alternative strategy is drug repurposing, which could identify FDA-approved and well-characterized drugs that could have off-label utility in treating SCV. In this study, we adapted a high-throughput AK-based assay to identify 4 FDA-approved drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, which display antimicrobial activity against S. aureus SCV, suggesting an avenue for drug repurposing in order to effectively treat SCV-related infections. Additionally, this screening paradigm can easily be adapted for other drug/chemical libraries to identify compounds bactericidal against SCV.

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca++ ] is consistent with parasite killing specifically via action on one or more Ca++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca++ transport and may be starting points for new antimalarial drugs.

Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.

Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.

Development and Therapeutic Potential of Small-Molecule Modulators of Circadian Systems.

Circadian timekeeping systems drive oscillatory gene expression to regulate essential cellular and physiological processes. When the systems are perturbed, pathological consequences ensue and disease risks rise. A growing number of small-molecule modulators have been reported to target circadian systems. Such small molecules, identified via high-throughput screening or derivatized from known scaffolds, have shown promise as drug candidates to improve biological timing and physiological outputs in disease models. In this review, we first briefly describe the circadian system, including the core oscillator and the cellular networks. Research progress on clock-modulating small molecules is presented, focusing on development strategies and biological efficacies. We highlight the therapeutic potential of small molecules in clock-related pathologies, including jet lag and shiftwork; various chronic diseases, particularly metabolic disease; and aging. Emerging opportunities to identify and exploit clock modulators as novel therapeutic agents are discussed.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM.

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.