High-Content Live-Cell Multiplex Screen for Chemogenomic Compound Annotation; Donated Chemical Probes
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ORGANISM(S): Homo sapiens (human)
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PROVIDER: S-BIAD568 | bioimages |
REPOSITORIES: bioimages
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Project description:BackgroundMalaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS) to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes.ResultsAfter screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells.ConclusionThis simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.
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Project description:High-content screening to monitor disease-modifying phenotypes upon small-molecule addition has become an essential component of many drug and target discovery platforms. One of the most common phenotypic approaches, especially in the field of oncology research, is the assessment of cell viability. However, frequently used viability readouts employing metabolic proxy assays based on homogeneous colorimetric/fluorescent reagents are one-dimensional, provide limited information, and can in many cases yield conflicting or difficult-to-interpret results, leading to misinterpretation of data and wasted resources.The resurgence of high-content, phenotypic screening has significantly improved the quality and breadth of cell viability data, which can be obtained at the very earliest stages of drug and target discovery. Here, we describe a relatively inexpensive, high-throughput, high-content, multiparametric, fluorescent imaging protocol using a live-cell method of three fluorescent probes (Hoechst, Yo-Pro-3, and annexin V), that is amenable to the addition of further fluorophores. The protocol enables the accurate description and profiling of multiple cell death mechanisms, including apoptosis and necrosis, as well as accurate determination of compound IC50, and has been validated on a range of high-content imagers and image analysis software. To validate the protocol, we have used a small library of approximately 200 narrow-spectrum kinase inhibitors and clinically approved drugs. This fully developed, easy-to-use pipeline has subsequently been implemented in several academic screening facilities, yielding fast, flexible, and rich cell viability data for a range of early-stage high-throughput drug and target discovery programs.
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Project description:PurposeThe combination of cytotoxic chemotherapy with radiation therapy (CRT) has resulted in significant improvements in clinical outcomes for patients with many locally advanced unresectable cancers. Only a small proportion of patients achieve pathologic complete responses to CRT; combination of CRT with targeted agents offers the promise of further improving treatment responses. However, numerous clinical trials have failed to show an improvement in clinical outcomes with the addition of targeted agents. To increase the accessibility of our screening method and accelerate the pace at which novel combinations with CRT are identified and incorporated into standard practices for treatments, we report details on screening method optimization, data generation, and downstream data analysis.MethodsIn part, the gap in translation to large, expensive, and ultimately unsuccessful clinical trials reflects the shortcomings of inconsistently designed, executed, and reported preclinical data on which these studies are based. In an effort to standardize the selection of agents for future clinical testing, we have designed, optimized and validated a high throughput, high content, clonogenic assay platform for step-wise progression of preclinical studies from in vitro to in vivo in non-small cell lung cancer and pancreatic ductal adenocarcinoma.ResultsThis highly stable in vitro method was standardized for identification of the most promising CTEP drugs that could best be combined with CRT from among as screen of multiple agents tested in an unbiased manner using 96-well plates. The methodology lends itself to seamless testing of multiple agents in a similar fashion allowing cross-comparisons, evaluation of CRT, or radiation therapy alone, and testing multiple concentrations of test agents sequenced at different times before and after radiation. The method identified Trametinib as a strong CRT sensitizer in KRAS-mutant non-small cell lung cancer and pancreatic ductal adenocarcinoma cell lines. This platform has enabled the screening and identification of several chemoradiation sensitizers.ConclusionsHigh throughput, high content clonogenic drug screening assay allows for the rapid identification of targets and agents to be translated to the clinic to help improve the effectiveness of current standard of care CRT in various solid tumors.
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