Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies

Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

A high-throughput fluorescent indicator displacement assay and principal component/cluster data analysis for determination of ligand-nucleic acid structural selectivity

ABSTRACTWe describe a high-throughput fluorescence indicator displacement assay (HT-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI diversity set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this HT-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

Real-Time Apoptosis and Viability High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer

Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development.

Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products

ABSTRACT The dormant phenotype acquired by Mycobacterium tuberculosis during infection poses a major challenge in disease treatment, since these bacilli show tolerance to front-line drugs. Therefore, it is imperative to find novel compounds that effectively kill dormant bacteria. By screening 4,400 marine natural product samples against dual-fluorescent M. tuberculosis under both replicating and nonreplicating conditions, we have identified compounds that are selectively active against dormant M. tuberculosis. This validates our strategy of screening all compounds in both assays as opposed to using the dormancy model as a secondary screen. Bioassay-guided deconvolution enabled the identification of unique pharmacophores active in each screening model. To confirm the activity of samples against dormant M. tuberculosis, we used a luciferase reporter assay and enumerated CFU. The structures of five purified active compounds were defined by nuclear magnetic resonance (NMR) and mass spectrometry. We identified two lipid compounds with potent activity toward dormant and actively growing M. tuberculosis strains. One of these was commercially obtained and showed similar activity against M. tuberculosis in both screening models. Furthermore, puupehenone-like molecules were purified with potent and selective activity against dormant M. tuberculosis. In conclusion, we have identified and characterized antimycobacterial compounds from marine organisms with novel activity profiles which appear to target M. tuberculosis pathways that are conditionally essential for dormancy survival.

Detour

This chapter argues that rear-screen projection in Edgar Ulmer's Detour (1945) is just as critical to the film's audiovisual economy as voiceover and flashback. Not unlike the radiophonic “theater of the mind” projected by the 1940s noir sound track, rear-screen projection acts as a secondary screen for the protagonist's psyche. In Detour, this oneiric screen, in addition to mobilizing two of the dominant affective modalities of classic noir—claustrophobia and phantasmagoria—operates as a temporal signpost. The result is that even as Al Roberts (Tom Neal), driven by the romance of the open road, strikes out for California, the back-screen projection is a constant reminder that the past can rear up at any moment and dash his dreams.

High Rates of Anal High-Grade Squamous Intraepithelial Lesions in HIV-Infected Women Who Do Not Meet Screening Guidelines

Background Human immunodeficiency virus (HIV)–infected women have a higher burden of anal high-grade squamous intraepithelial lesions (HSIL) and anal cancer (AC) compared with HIV-uninfected women. Guidelines for AC screening in this population are heterogeneous. Here we report outcomes and risk factors for anal HSIL following implementation of universal AC screening offered to all HIV-infected women. Methods Data from women who underwent AC screening with anal cytology from April 2009 to July 2014 were analyzed. Routine clinical data included anal and cervical cytology, demographic/behavioral data, and high-resolution anoscopy (HRA) results. We evaluated the association of cytology with HRA results, and predictors of HSIL pathology, and compared rates of HSIL pathology among women meeting screening guidelines to those who did not. Results Seven hundred forty-five HIV-infected women were screened with anal cytology. Thirty-nine percent had abnormal anal cytology on initial screen and 15% on secondary screen; 208 women underwent HRA following abnormal anal cytology. HSIL was found in 26% and 18% of anal biopsies following initial and secondary screening, respectively. One woman had AC. Cigarette smoking more than doubled HSIL risk. Among women who underwent AC screening despite not meeting existing guideline criteria, 21% and 10%, respectively, were found to have HSIL on biopsy. Neither meeting criteria for screening nor history of receptive anal sex was significantly associated with HSIL. Conclusions Anal HSIL is common in HIV-infected women. Substantial numbers of HSIL would have been missed by strictly adhering to existing AC screening guidelines. These results support routine screening of all HIV-infected women regardless of human papillomavirus history or sexual practices.

Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs

Drosophila larval crawling is an attractive system to study patterned motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors including head casts, turning, and feeding. It is likely that some neurons are used in all these behaviors (e.g. motor neurons), but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines – chosen for sparse neuronal expression – to express the warmth-inducible neuronal activator TrpA1 and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°C). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program.