Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99

Lemieux, Benoit; Percival, M. David; Falgueyret, Jean‐Pierre

doi:10.1016/j.ab.2004.01.010

Cited by 88 publications

(93 citation statements)

References 8 publications

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“…S3C) or significantly affect viability at autophagymodulating doses (<10% effect; SI Appendix, Fig. S3D) (32). These data suggest that BRD5631 and other hits do not increase GFP-LC3 punctae formation by broadly disrupting lysosomal function.…”

Section: Resultsmentioning

confidence: 60%

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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Section: Resultsmentioning

confidence: 60%

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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“…We next studied agonist-stimulated trafficking of S1P 1 -GFP by confocal microscopy, using two cytoplasmic vesicle markers: the mannose 6-phosphate (M6P) receptor, which associates with late endosomes (21), and Lysotracker Red, a pH-sensitive dye, which labels lysosomes (22). Two time points were chosen, a 1-h protocol, which corresponded to maximal agonist-induced ubiquitin receptor complex, and 4 h, where agonist differences in down-regulation were first noticed.…”

Section: Resultsmentioning

confidence: 99%

Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics

Gonzalez-Cabrera

Hla

Rosen

2007

Journal of Biological Chemistry

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Sphingosine 1-phosphate subtype 1 (S1P 1 ) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P 1 down-regulation. Discovery of the in vivo active S1P 1 -selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P 1 function while highlighting differences regarding agonist effect on S1P 1 fate, because SEW2871 does not degrade S1P 1 . To further understand the mechanism of agonist-induced S1P 1 down-regulation, we compared signaling and fate of human S1P 1 -green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P 1 to late endosomal vesicles and activated GTP␥S 35 binding and pERK to similar maxima, only AFD-R led to significant S1P 1 down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time-and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P 1 . All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P 1 and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P 1 (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.Trafficking of agonist-stimulated G protein-coupled receptors (GPCRs) 2 classically proceeds through time-dependent steps starting by acute (within minutes) internalization of receptors from plasma membrane into cytoplasmic vesicular compartments and followed by receptor sorting into either recycling or degradative pathways, which usually take place within hours of agonist treatment. The long term effects of agonists on GPCR fate are dependent on both the nature of the agonist as well as the cellular environment and are believed to be important determinants of agonist effectiveness. Several mechanisms implicated in early GPCR trafficking have been described and involve phosphorylation of agonist-stimulated receptor by GPCR kinases and subsequent binding of arrestin proteins to phosphorylated receptor sites and to adapter proteins such as clathrin and AP-2, which form coated-pit receptor complexes (reviewed in Refs. 1 and 2). Based on the stability of the arrestin-receptor complexes, GPCRs have been separated into class A receptors, which favor the recycling pathway, and class B receptors, which recycle slowly and are instead destined for degradation ...

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“…Such molecules may accumulate in lysosomes. For example, accumulation of propranolol has been observed (Ishizaki et al 2000, Lemieux et al 2004). Propranolol has a basic group with pK a 9.14 and an acidic OH group with pK a at 13.84 (ACD 2007).…”

Section: Bivalent Bases (Z = +2)mentioning

confidence: 99%

Quantitative modeling of selective lysosomal targeting for drug design

et al. 2008

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Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick-Nernst-Planck-equation. The cell model considers the diffusion of neutral and ionic molecules across biomembranes, dissociation to mono-or bivalent ions, adsorption to lipids, and electrical attraction or repulsion. Based on simulation results, high and selective accumulation in lysosomes was found for weak mono-and bivalent bases with intermediate to high log Kow. These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the optimum range predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation. This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions.

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Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99

Cited by 88 publications

References 8 publications

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

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

Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics

Quantitative modeling of selective lysosomal targeting for drug design

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