A high content screening assay for identifying lysosomotropic compounds

Nadanaciva, Sashi; Lu, Shuyan; Gebhard, David F.; Jessen, Bart; Pennie, William D.; Will, Yvonne

doi:10.1016/j.tiv.2010.12.010

Cited by 186 publications

(181 citation statements)

References 31 publications

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“…To demonstrate that the weak base imatinib is sequestered in acidic lysosomes, we coincubated H226 cells with imatinib and LysoTracker Red DND-99, a viable fluorophore readily entering acidic subcellular organelles such as lysosomes (Nadanaciva et al, 2011). Confocal imaging clearly showed that the red fluorescent pattern from LysoTracker Red colocalized with the imatinib-associated green fluorescent signal as indicated by the yellow color in the overlay (Fig.…”

Section: Resultsmentioning

confidence: 97%

Lysosomal Sequestration Determines Intracellular Imatinib Levels

et al. 2015

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The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH 4 Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression.

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Section: Resultsmentioning

confidence: 97%

Lysosomal Sequestration Determines Intracellular Imatinib Levels

et al. 2015

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“…Only analogs containing an amine retained activity, and both removal of the terminal phenyl group and its replacement with a pyridyl group eliminated activity of BRD5631. Compounds containing a lipophilic group and a basic amine can accumulate within lysosomes (lysosomotropism) and potentially interfere with lysosomal function, cell viability, and autophagy (29,30). To determine whether our hits disrupt lysosomal function, we first tested their effect on lysosomal proteases by measuring processing of a fluorogenic substrate (DQ-BSA) (31).…”

Section: Resultsmentioning

confidence: 99%

Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics

Kuo

Castoreno

Aldrich

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.high-throughput screening | autophagy | small-molecule probes | Crohn's disease

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“…For example amodiaquine and chloroquine are well known antimalarials, clomiphene and toremifene are selective estrogen receptor modulators, while amiodarone, dronedarone, and verapamil are anti-arrhythmics 4 . It may or may not be of importance but all of these compounds have a common tertiary amine feature 10, 11 . What is important is that they are all orally bioavailable and generally safe for humans at their approved doses.…”

Section: Introductionmentioning

confidence: 99%

Machine learning models identify molecules active against the Ebola virus in vitro

et al. 2016

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The search for small molecule inhibitors of Ebola virus (EBOV) has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs) with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC 50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in vitro.

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A high content screening assay for identifying lysosomotropic compounds

Cited by 186 publications

References 31 publications

Lysosomal Sequestration Determines Intracellular Imatinib Levels

Lysosomal Sequestration Determines Intracellular Imatinib Levels

Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics

Machine learning models identify molecules active against the Ebola virus in vitro

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