| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
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| Targets |
TRPML1 (IC50 = 1.6 μM); TRPML2 (IC50 = 2.3 μM); TRPML3 (IC50 = 12.5 μM)
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| ln Vitro |
HeLa cells' ML-SA1-induced Ca2+ signaling is inhibited by ML-SI3 (10 μM) [2]. Adult schistosoma membrane integrity is disrupted by ML-SI3 (25-75 μM, 24 hours) [3]. In the modeled lysosomal lumen, rapamycin-induced ITRPML1 is blocked by ML-SI3 (10 μM) [4]. In newborn rat ventricular myocytes (NRVM), ML-SI3 (3 µM, 6 h) completely eliminates the increases in LC3II and p62 levels that are caused by hypoxia/reoxygenation (H/R) (4 h H/2 h R) [5].
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| ln Vivo |
ML-SI3 can lessen I/R damage in mouse cardiomyocytes when injected intraperitoneally four times at a dose of 1.5 mg/kg [5].
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| Enzyme Assay |
Concentration-effect measurements were based on a Fluo-4/AM assay and were performed by using a custom-made fluorescence imaging plate reader (FLIPR) built into a robotic liquid handling station. All imaging experiments were done in a HEPES buffered solution (HBS), containing 132 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.5 mM d-glucose, 10 mM HEPES, pH 7.4. Compounds dissolved in DMSO (10 mM) were serially prediluted in HBS (0.98 μM-1 mM). HEK293 cells stably expressing plasma membrane-targeted human TRPML1, TRPML2 or TRPML3 [14] were trypsinized and resuspended in cell culture medium supplemented with 4 μM Fluo-4/AM. After incubation at 37 °C for 30 min, the cell suspension was briefly centrifuged, resuspended in HBS and dispensed into black pigmented, clear-bottom 384-well microwell plates. Then plates were placed into the FLIPR and fluorescence signals (excitation 470 nm, emission 515 nm) were recorded with a Zyla 5.5 camera nd the μManager software like previously described. In a first step and video, theTecan 96-tip multichannel arm added a negative HBS control or the prediluted compounds to the cells in final concentrations of 0.098 μM–100 μM. To map antagonistic effects, ML-SA1 (5 μM) was subsequently pipetted in each well and fluorescence signals were recorded for 10 min. Analyses were performed by calculating fluorescence intensities for each well and background areas with ImageJ. Finally, the background was subtracted and the fluorescence intensities were normalized to initial intensities (F/F0). For comparing inhibition potency of compounds, a second normalization to the negative control was done. All concentration-effect curves were fitted to a four-parameter Hill equation to obtain Imin, Imax, IC50) and the Hill coefficient n.[2]
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| Cell Assay |
Single cell Ca2+ imaging experiments were performed using Fura-2 as previously described. HEK293 cells stably expressing hTRPML1ΔNC-YFP, hTRPML2-YFP or hTPPML3-YFP were cultured at 37 °C with 5% of CO2 in Dulbecco’s modified Eagle medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were plated onto poly-l-lysine (sigma)-coated glass coverslips and grown for 2–3 days. For Ca2+ imaging experiments cells were loaded for 45 min at 37 °C with Fura-2 AM (4.0 μM) and 0.005% (v/v) pluronic acid in HEPES-buffered solution (HBS) comprising 138 mM NaCl, 6 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES and 5.5 mM d-glucose (adjusted to pH 7.4 with NaOH). After loading, cells were washed with HBS and mounted in an imaging chamber. Experiments were carried out as previously described. After stimulation with an activator (10 μM) for 200 s, the inhibitor (10 μM) was applied for another 200 s. Activation was normalized to 1. All recordings were performed in HBS on a Leica DMi8 live cell microscope or a Polychrome IV mono-chromator (only for experiments with transiently transfected hTRPML1 HEK293 cells). Fura-2 was excited at 340 nm/380 nm. Emitted fluorescence was captured using 515 nm long-pass filter. Compounds were prediluted in DMSO and stored as 10 mM stock solutions at −20 °C, not exceeding three months. Working solutions were prepared directly before using by dilution with HBS. In all statistical analyses of Ca2+ imaging experiments, mean values of at least three independent experiments are shown as indicated. ∗∗∗ indicates p < 0.001, ∗∗ indicates p < 0.01, ∗ indicates p < 0.05, ns = not significant, one-way ANOVA test followed by Tukey’s post-hoc test.
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| Animal Protocol |
Animal/Disease Models: Myocardial ischemia/reperfusion (I/R) injury in mice [5]
Doses: 1.5 mg/kg Route of Administration: intraperitoneal (ip) injection, four times before and during in vivo I/R (30 minutes of ischemia , 1 day of reperfusion) ) Experimental Results: Blocked autophagic flux in I/R cardiomyocytes was restored. |
| References |
[1]. Rühl P, et al. Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors. Sci Rep. 2021 Apr 15;11(1):8313.
[2]. Leser C, et al. Chemical and pharmacological characterization of the TRPML calcium channel blockers ML-SI1 and ML-SI3. Eur J Med Chem. 2021 Jan 15;210:112966. [3]. Kilpatrick BS, et al. Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx. J Cell Sci. 2016 Oct 15;129(20):3859-3867. [4]. Bais S, et al. Schistosome TRPML channels play a role in neuromuscular activity and tegumental integrity. Biochimie. 2022 Mar;194:108-117. [5]. Zhang X, et al. Rapamycin directly activates lysosomal mucolipin TRP channels independent of mTOR. PLoS Biol. 2019 May 21;17(5):e3000252. [6]. Xing Y, et al. Blunting TRPML1 channels protects myocardial ischemia/reperfusion injury by restoring impaired cardiomyocyte autophagy. Basic Res Cardiol. 2022 Apr 7;117(1):20. |
| Additional Infomation |
Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.[5]
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| Molecular Formula |
C23H31N3O3S
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|---|---|
| Molecular Weight |
429.58
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| Exact Mass |
429.21
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| Elemental Analysis |
C, 64.31; H, 7.27; N, 9.78; O, 11.17; S, 7.46
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| CAS # |
2418594-00-8
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| Related CAS # |
(1S,2S)-ML-SI3;2563870-87-9;(rel)-ML-SI3;2108567-79-7;ML-SI3;891016-02-7
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| PubChem CID |
94784696
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
589.3±60.0 °C at 760 mmHg
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| Flash Point |
310.2±32.9 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.629
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| LogP |
4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
624
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| Defined Atom Stereocenter Count |
2
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| SMILES |
COC1=CC=CC=C1N2CCN(CC2)[C@@H]3CCCC[C@H]3NS(=O)(=O)C4=CC=CC=C4
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| InChi Key |
OVTXOMMQHRIKGL-NHCUHLMSSA-N
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| InChi Code |
InChI=1S/C23H31N3O3S/c1-29-23-14-8-7-13-22(23)26-17-15-25(16-18-26)21-12-6-5-11-20(21)24-30(27,28)19-9-3-2-4-10-19/h2-4,7-10,13-14,20-21,24H,5-6,11-12,15-18H2,1H3/t20-,21-/m1/s1
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| Chemical Name |
N-[(1R,2R)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide
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| Synonyms |
(1R,2R)-ML-SI3; rel-N-((1R,2R)-2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide; CHEMBL4851704; N-((1R,2R)-2-(4-(2-methoxyphenyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide; 2418594-00-8; N-[(1R,2R)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide; (rel)-ML-SI3;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~232.79 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3279 mL | 11.6393 mL | 23.2786 mL | |
| 5 mM | 0.4656 mL | 2.3279 mL | 4.6557 mL | |
| 10 mM | 0.2328 mL | 1.1639 mL | 2.3279 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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