“… Figure 4 Estradiol, EDME and analogs in Fluo-4 calcium imaging experiments. ( a – l ) Concentration-effect relationships for Ca 2+ increases (Fluo-4) in response to different concentrations of EDME, estradiol and analogs on HEK293 cells stably expressing hTRPML1∆NC-YFP, hTRPML2-YFP, hTRPML3-YFP or hTPC2L11A/L12A-RFP 22 , 24 . Cells were activated with ML-SA1 (5 µM) for TRPMLs or TPC2-A1-N (10 µM; blue) and TPC2-A1-P (30 µM; orange) for hTPC2.…”
Section: Resultsmentioning
confidence: 99%
“…Only inhibitors without isoform-selectivity, e.g. ML-SI1 and ML-SI3 have been described so far 23 , 24 . TRPML channel isoforms can occur not only in the same cell type, e.g.…”
The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17β-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.
“… Figure 4 Estradiol, EDME and analogs in Fluo-4 calcium imaging experiments. ( a – l ) Concentration-effect relationships for Ca 2+ increases (Fluo-4) in response to different concentrations of EDME, estradiol and analogs on HEK293 cells stably expressing hTRPML1∆NC-YFP, hTRPML2-YFP, hTRPML3-YFP or hTPC2L11A/L12A-RFP 22 , 24 . Cells were activated with ML-SA1 (5 µM) for TRPMLs or TPC2-A1-N (10 µM; blue) and TPC2-A1-P (30 µM; orange) for hTPC2.…”
Section: Resultsmentioning
confidence: 99%
“…Only inhibitors without isoform-selectivity, e.g. ML-SI1 and ML-SI3 have been described so far 23 , 24 . TRPML channel isoforms can occur not only in the same cell type, e.g.…”
The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17β-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.
“…Recent reports involving ML-SI3 and a related agonist ML-SA5 further exacerbate these complexities by revealing that ML-SI3 may have enantiomerspecific activities. For TRPML1, the (+)-and (À)-enantiomers have varying levels of inhibition, while for TRPML2 and TRPML3, the (+)-enantiomer reverses its effect, displaying weak stimulatory properties (Leser et al, 2021). More physiologically relevant, however, is the way in which ML-SI3 does not impact the stimulatory effect of PI(3,5)P 2 (Figure 3F), despite the synergistic increase in TRPML1 activity and open probability observed with both ML-SA1 and PI(3,5)P 2 (Chen et al, 2017).…”
Section: Discussionmentioning
confidence: 99%
“…Earlier reports show effective stimulation of TRPML1 inward rectifying cation currents at a working ML-SA1 concentration in the low-micromolar range. However, the values are highly dependent on methodology (i.e., Ca 2+ fluorescence, planar bilayer recordings, and lysosomal patch clamp) with limited demonstration of the concentration ranges required for wholecell electrophysiology (Dong et al, 2008;Leser et al, 2021). Environmental factors (i.e., extracellular pH and cationic constituents) also contribute to the range of activities reported for TRPML agonists (Dong et al, 2008;Li et al, 2017).…”
Highlights d A 2.9Å cryo-EM structure of ML-SI3-bound TRPML1 reveals a closed conformation d The TRPML1 inhibitor ML-SI3 binds the same cavity as its agonist ML-SA1 d ML-SI3 competes with ML-SA1 to block the channel activity d TRPML1 inactivation via ML-SI3 is independent of phosphoinositide regulation
“…These findings indicate that activating TRPML1 could be a promising strategy to induce autophagy and promote the clearance of α-synuclein aggregates. To confirm this hypothesis, we used the cell permeable Mucolipin-synthetic agonist (ML-SA1) to specifically and potently activate TRPML-1 (Shen et al, 2012 ), and the mucolipin-synthetic antagonist (ML-SI3) to block TRPML1 (Leser et al, 2021 ). We expressed the disease-related A53T mutant of α-synuclein in HEK293T cells and determined whether treatment with ML-SA1 affects the amount of A53T-α-synuclein protein.…”
Background: Protein aggregates are degraded via the autophagy-lysosome pathway and alterations in the lysosomal system leading to the accumulation of pathogenic proteins, including aggregates of α-synuclein in Parkinson’s disease (PD). The importance of the endolysosomal transient receptor potential cation channel, mucolipin subfamily 1 (TRPML1) for the lysosomal function is highlighted by the fact that TRPML1 mutations cause the lysosomal storage disease mucolipidosis type IV. In this study, we investigated the mechanism by which activation of TRPML1 affects the degradation of α-synuclein.Methods: As a model of α-synuclein pathology, we expressed the pathogenic A53Tα-synuclein mutant in HEK293T cells. These cells were treated with the synthetic TRPML1 agonist ML-SA1. The amount of α-synuclein protein was determined by immunoblots. The abundance of aggregates and autolysosomal vesicles was determined by fluorescence microscopy and immunocytochemistry. Findings were confirmed by life-cell imaging and by application of ML-SA1 and the TRPML1 antagonist ML-SI3 to human dopaminergic neurons and human stem cell-derived neurons.Results: ML-SA1 reduced the percentage of HEK293T cells with α-synuclein aggregates and the amount of α-synuclein protein. The effect of ML-SA1 was blocked by pharmacological and genetic inhibition of autophagy. Consistent with TRPML function, it required the membrane lipid PI(3,5)P2, and cytosolic calcium. ML-SA1 shifted the composition of autophagosomes towards a higher fraction of mature autolysosomes, also in presence of α-synuclein. In neurons, inhibition of TRPML1 by its antagonist ML-SI3 blocked autophagosomal clearance, whereas the agonist ML-SA1 shifted the composition of a-synuclein particles towards a higher fraction of acidified particles. ML-SA1 was able to override the effect of Bafilomycin A1, which blocks the fusion of the autophagosome and lysosome and its acidification.Conclusion: These findings suggest, that activating TRPML1 with ML-SA1 facilitates clearance of α-synuclein aggregates primarily by affecting the late steps of the autophagy, i.e., by promoting autophagosome maturation. In agreement with recent work by others, our findings indicate that TRPML1 might constitute a plausible therapeutic target for PD, that warrants further validation in rodent models of α-synuclein pathology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
