High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs

Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping

LCIO: A persistency framework and event data model for HEP

Abstract Not Provide

HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3’UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG5) and DM1 patient (DMPK CTG1000) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 μM; and N. fowleri EC50: 0.43 ± 0.13 μM), 1c and 2b (N. fowleri EC50s: <0.63 μM, and 0.3 ± 0.21 μM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 μM, and 1.4 ± 0.17 μM, respectively). With several of these pharmacophores already possessing blood–brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases

An exploratory study of the relationship between parental attitudes and behaviour and young people's consumption of alcohol

Background Concern is growing regarding frequent and excessive misuse of alcohol by young people. The average age at which young people in Europe start to drink is twelve and a half, and during the last decade, the quantity of alcohol consumed by younger adolescents in the UK has increased. Families are known to play an important role in shaping young people's alcohol misuse, although family risk and protective factors associated with misuse in a UK context are in need of further investigation. Methods The study used a cross-sectional design, involving secondary analyses of self-completion questionnaire responses from 6,628 secondary school children (i.e. aged 11-16 years), from 12 schools within an urban location in Wales. Items relating to family functioning and perceived parental attitudes were first subjected to factor analysis. Associations of family closeness and conflict, parental monitoring and attitudes and family history of substance misuse with children's self reported alcohol consumption were examined using logistic regression analyses. Results Approximately three quarters of respondents reported having tried alcohol, most of whom had first tried alcohol aged 12 or under. Parental monitoring and family closeness were positively correlated with one another and were both associated with significantly lower levels of drinking behaviours. Family violence and conflict, more liberal parental attitudes towards substance use and towards alcohol and petty crime, and family history of substance misuse were positively correlated with one another and with higher levels of drinking behaviours. Parental monitoring was identified as the family functioning factor most consistently associated with drinking behaviour in multivariate analyses. Conclusions Significant relationships were found between young people's drinking behaviours and perceptions of risk and protective factors in the family environment. Parental monitoring was strongly associated with family closeness and appeared to form one part of a parenting style of more general communication and regulation of children's behaviour. Findings support the need for alcohol misuse prevention interventions which address risk and protective factors within the family setting. Timing of such prevention work should be related both to the development of family relationships and the age at which young people begin drinking alcohol

Modern American populism: Analyzing the economics behind the Silent Majority, the Tea Party and Trumpism

This article researches populism, more specifically, Modern American Populism (MAP), constructed of white, rural, and economically oppressed reactionarianism, which was borne out of the political upheaval of the 1960’s Civil Rights movement. The research looks to explain the causes of populism and what leads voters to support populist movements and politicians. The research focuses on economic anxiety as the main cause but also examines an alternative theory of racial resentment. In an effort to answer the question, what causes populist movements and motivations, I apply a research approach that utilizes qualitative and quantitative methods. There is an examination of literature that defines populism, its causes and a detailed discussion of the case studies, including the 1972 election of Richard Nixon; the Tea Party election of 2010; and the 2016 election of Donald Trump. In addition, statistical data analysis was run using American National Election Studies (ANES) surveys associated with each specific case study. These case studies were chosen because they most represent forms of populist movements in modern American history. While ample qualitative evidence suggested support for the hypothesis that economic anxiety is a necessary condition for populist voting patterns that elected Nixon, the Tea Party and Trump, the statistical data only supported the hypothesis in two cases, 2010 and 2016, with 1972 coming back inconclusive. The data also suggested that both economic anxiety and racial resentment played a role in 2010 and 2016, while having no significant effect in 1972 in either case. This suggests that further research needs to be conducted into additional populist case studies, as well as an examination into the role economic anxiety and economic crises play on racial resentment and racially motivated voting behavior