Lysosomal Sequestration (Trapping) of Lipophilic Amine (Cationic Amphiphilic) Drugs in Immortalized Human Hepatocytes (Fa2N-4 Cells)

Kazmi, Faraz; Hensley, Tiffini; Pope, Chad; Funk, Ryan S.; Loewen, Greg; Buckley, David B.; Parkinson, Andrew

doi:10.1124/dmd.112.050054

Cited by 208 publications

(214 citation statements)

References 35 publications

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“…Almost all (95%) of Gleevec entering cells (regardless of cell type) becomes sequestered in lysosomes (25,26) through ion trapping, because Gleevec is a weak base (27)(28)(29). To examine the possibility that Gleevec might inhibit the activities of lysosomal proteases by raising lysosomal pH, we exposed N2a 695 cells to vehicle or to Gleevec for 4 h and then to the lysosomotropic dye Lysotracker Red (Thermo Fisher Scientific), which sequesters in acidified lysosomes (30).…”

Section: Resultsmentioning

confidence: 99%

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

Netzer

Bettayeb

Sinha

et al. 2017

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

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Neurotoxic amyloid-β peptides (Aβ) are major drivers of Alzheimer's disease (AD) and are formed by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE) and γ-secretase. Our previous study showed that the anticancer drug Gleevec lowers Aβ levels through indirect inhibition of γ-secretase activity. Here we report that Gleevec also achieves its Aβ-lowering effects through an additional cellular mechanism. It renders APP less susceptible to proteolysis by BACE without inhibiting BACE enzymatic activity or the processing of other BACE substrates. This effect closely mimics the phenotype of APP A673T, a recently discovered mutation that protects carriers against AD and age-related cognitive decline. In addition, Gleevec induces formation of a specific set of APP C-terminal fragments, also observed in cells expressing the APP protective mutation and in cells exposed to a conventional BACE inhibitor. These Gleevec phenotypes require an intracellular acidic pH and are independent of tyrosine kinase inhibition, given that a related compound lacking tyrosine kinase inhibitory activity, DV2-103, exerts similar effects on APP metabolism. In addition, DV2-103 accumulates at high concentrations in the rodent brain, where it rapidly lowers Aβ levels. This study suggests that long-term treatment with drugs that indirectly modulate BACE processing of APP but spare other BACE substrates and achieve therapeutic concentrations in the brain might be effective in preventing or delaying the onset of AD and could be safer than nonselective BACE inhibitor drugs.Alzheimer's disease | Gleevec | nonamyloidogenic | β-cleavage

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Section: Resultsmentioning

confidence: 99%

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

Netzer

Bettayeb

Sinha

et al. 2017

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

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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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“…This indicated that VP35 may be a potential target for these two distinct classes of compounds. However, it is important to point out that we have not compared docking to other proteins in EBOV and it could also be possible that these molecules are active elsewhere as well as via other mechanisms than by specific binding to proteins 28, 29 . Further, VP35 may be a preferred target for the antimalarials while the SERMs are not predicted to bind as well as the X-ray ligand.…”

Section: Discussionmentioning

confidence: 99%

A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus

2014

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We are currently faced with a global infectious disease crisis which has been anticipated for decades. While many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the Ebola or similar filoviruses that cause haemorrhagic fever. Two recent high throughput screens published in 2013 did however identify several hits that progressed to animal studies that are FDA approved drugs used for other indications. The current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore. This ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored. A recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein 35 (VP35). When receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on FDA approved drugs. These previously identified FDA approved drugs with activity against Ebola were therefore docked into this protein. The antimalarials chloroquine and amodiaquine docked favorably in VP35. We propose that these drugs identified to date as inhibitors of the Ebola virus may be targeting VP35. These computational models may provide preliminary insights into the molecular features that are responsible for their activity against Ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested.

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Lysosomal Sequestration (Trapping) of Lipophilic Amine (Cationic Amphiphilic) Drugs in Immortalized Human Hepatocytes (Fa2N-4 Cells)

Cited by 208 publications

References 35 publications

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

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

A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus

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