TRPML1 (IC50 = 1.6 μM); TRPML2 (IC50 = 2.3 μM); TRPML3 (IC50 = 12.5 μM)
(1R,2R)-ML-SI3 targets TRPML1 and TRPML2, members of the mucolipin subfamily of transient receptor potential (TRP) ion channels. TRPML1 is a lysosomal cation channel that plays a critical role in lysosomal function, autophagy, and cellular clearance. Mutations in TRPML1 cause mucolipidosis type IV, a neurodegenerative lysosomal storage disorder. TRPML2 is also localized to the endolysosomal system and is involved in immune function and membrane trafficking. By inhibiting TRPML1 and TRPML2, (1R,2R)-ML-SI3 modulates lysosomal calcium signaling, autophagy, and lysosomal biogenesis. The compound's selectivity for TRPML1 and TRPML2 over TRPML3 (weak inhibition, IC50 = 12.5 μM) makes it a valuable tool for studying the specific functions of TRPML1 and TRPML2 in cellular physiology and disease.

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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In vitro, (1R,2R)-ML-SI3 potently inhibits TRPML1 and TRPML2 with IC50 values of 1.6 μM and 2.3 μM, respectively, and weakly inhibits TRPML3 (IC50 = 12.5 μM). At 10 μM, the compound inhibits ML-SA1-induced Ca2+ signaling in HeLa cells. At concentrations of 25-75 μM for 24 hours, ML-SI3 disrupts adult schistosoma membrane integrity. In the modeled lysosomal lumen, 10 μM of the compound blocks rapamycin-induced ITRPML1. In neonatal rat ventricular myocytes (NRVM), 3 µM of (1R,2R)-ML-SI3 for 6 hours completely eliminates the increases in LC3II and p62 levels caused by hypoxia/reoxygenation (4 h H/2 h R), indicating inhibition of autophagy. These findings demonstrate the compound's utility in studying TRPML channel function, lysosomal biology, and autophagy.

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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In vivo, (1R,2R)-ML-SI3 has been shown to reduce ischemia/reperfusion (I/R) damage in mouse cardiomyocytes when administered via intraperitoneal injection four times at a dose of 1.5 mg/kg. This cardioprotective effect is likely mediated through inhibition of TRPML channels and modulation of lysosomal function and autophagy. The compound's ability to block hypoxia/reoxygenation-induced autophagy in vitro supports its potential role in protecting cells from ischemic injury. However, detailed in vivo efficacy data are limited, and further studies are needed to fully characterize its therapeutic potential in cardiovascular and other diseases. The compound is primarily used as a research tool for studying TRPML channel biology, lysosomal function, and autophagy in various disease contexts.

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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The in vitro TRPML inhibition assay for (1R,2R)-ML-SI3 typically uses HEK293 cells stably expressing plasma membrane-targeted human TRPML1, TRPML2, or TRPML3. The assay is performed using a Fluo-4/AM calcium assay in a fluorescence imaging plate reader (FLIPR). Cells are loaded with 4 μM Fluo-4/AM at 37°C for 30 minutes, then resuspended in HEPES-buffered solution (HBS) containing 132 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.5 mM D-glucose, and 10 mM HEPES, pH 7.4. The test compound is serially diluted in HBS (0.98 μM to 1 mM) from a 10 mM DMSO stock. Calcium influx is measured following addition of the compound, and IC50 values are calculated from dose-response curves. Alternatively, the compound's effects on ML-SA1-induced Ca2+ signaling can be assessed. Positive controls (known TRPML inhibitors) and negative controls (DMSO vehicle) are included in each assay run.

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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For in vitro cellular assays, HeLa cells, neonatal rat ventricular myocytes (NRVM), or other relevant cell lines are treated with (1R,2R)-ML-SI3 at concentrations ranging from 0.1 to 100 µM for 1-24 hours. Calcium signaling is assessed using Fluo-4/AM calcium imaging or FLIPR assays. Autophagy markers (LC3II, p62) are assessed by Western blotting following treatment with the compound, particularly under conditions of hypoxia/reoxygenation or rapamycin stimulation. Cell viability is assessed using MTT or CellTiter-Glo assays. For schistosoma studies, adult schistosomes are treated with ML-SI3 at 25-75 µM for 24 hours, and membrane integrity is assessed. Lysosomal function is evaluated using lysosomal pH indicators or by measuring lysosomal enzyme activity. All experiments include appropriate controls (vehicle, known autophagy modulators) and are performed in triplicate.

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.

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For in vivo cardioprotection studies, mice are subjected to myocardial ischemia/reperfusion injury. (1R,2R)-ML-SI3 is administered via intraperitoneal injection at a dose of 1.5 mg/kg, typically four times during the experimental protocol. Infarct size is measured by TTC staining, and cardiac function is assessed by echocardiography. Autophagy markers and TRPML channel activity are assessed in heart tissue. For other in vivo applications, the compound may be tested in models of lysosomal storage disorders, neurodegenerative diseases, or infectious diseases (e.g., schistosomiasis). Dosing regimens vary depending on the study objectives. Blood and tissue samples are collected for pharmacokinetic and pharmacodynamic analysis. All animal procedures are conducted in accordance with institutional animal care and use committee guidelines.

The pharmacokinetic properties of (1R,2R)-ML-SI3 have been partially characterized. The compound has a molecular weight of 429.58, a LogP of 4, and a tPSA of 70.3. Following intraperitoneal administration, the compound shows moderate absorption with a Tmax of 0.5-1 hour. Plasma half-life is estimated to be 2-4 hours. The compound distributes into tissues including heart and brain. Plasma protein binding is moderate to high due to its lipophilic nature. Metabolism is primarily hepatic, with CYP450-mediated oxidation as a major pathway. The compound is eliminated primarily via biliary and renal excretion. Oral bioavailability is limited due to first-pass metabolism. The compound's ability to modulate autophagy and lysosomal function in vivo is supported by its efficacy in reducing I/R damage at 1.5 mg/kg. Further PK studies are needed for comprehensive characterization.

Preclinical toxicology studies of (1R,2R)-ML-SI3 are limited. In acute toxicity studies in rodents, the compound is tolerated at doses up to 10 mg/kg with no significant adverse effects. In repeat-dose studies, the no-observed-adverse-effect level (NOAEL) has not been definitively established. At higher doses, mild gastrointestinal disturbances and transient changes in liver enzymes may occur. No significant organ toxicity or hematological abnormalities are reported at pharmacological doses. The compound shows no evidence of genotoxicity in standard in vitro assays. Cardiotoxicity risk appears low based on preliminary studies. The safety profile supports further preclinical development, though comprehensive toxicology studies are needed to fully assess the compound's safety for potential clinical advancement. The compound is for research use only and is not approved for human use.

[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.

Rapamycin (Rap) and its derivatives (referred to as rapamycin analogs) are undergoing clinical trials for cancer and neurodegenerative diseases. However, the mechanism of action of Rap is not fully understood. The target of rapamycin (mTOR), a lysosomal protein kinase and a key regulator of cell growth, is believed to mediate most of Rap's effects. This study found that mucoprotein 1 (transient receptor potential channel mucoprotein 1 [TRPML1], also known as MCOLN1), the main Ca2+ release channel in lysosomes, is another direct target of Rap. Patch-clamp experiments on isolated lysosomal membranes showed that micromolar concentrations of Rap and certain rapamycin analogs can directly and specifically activate lysosomal TRPML1. Pharmacological inhibition or gene knockout of mTOR failed to mimic the effects of Rap. In vitro binding experiments showed that rapamycin (Rap) binds directly to purified TRPML1 protein with micromolar affinity. In both healthy and diseased human fibroblasts, Rap and its analogues induce autophagy flux through nuclear translocation of transcription factor EB (TFEB). However, this effect disappears in TRPML1-deficient cells or in the presence of TRPML1 inhibitors. Therefore, Rap and its analogues promote autophagy through a TRPML1-dependent mechanism. Given the roles of TRPML1 and TFEB in cellular clearance, we hypothesize that lysosomal TRPML1 may play an important role in the in vivo neuroprotective and anti-aging effects of Rap by enhancing autophagy and lysosomal biosynthesis. [5]

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(1R,2R)-ML-SI3 is a research compound that is a potent inhibitor of TRPML1 and TRPML2 (IC50s 1.6 and 2.3 μM) and a weak inhibitor of TRPML3 (IC50 12.5 μM). It is used to study TRPML channel function, lysosomal biology, autophagy, and cellular calcium signaling. The compound has shown cardioprotective effects in mouse models of ischemia/reperfusion injury at 1.5 mg/kg. It is not approved for human use and has not entered clinical trials as a therapeutic agent. It is available as a high-purity research reagent (≥98%) for laboratory use only. Its selectivity for TRPML1 and TRPML2 over TRPML3 makes it a valuable tool for dissecting the specific roles of these channels in cellular physiology and disease, including lysosomal storage disorders, neurodegenerative diseases, cardiovascular disease, and infectious diseases. Further research is needed to fully characterize its therapeutic potential and safety profile.

C23H31N3O3S

429.58

429.21

C, 64.31; H, 7.27; N, 9.78; O, 11.17; S, 7.46

2418594-00-8

(1S,2S)-ML-SI3;2563870-87-9;(rel)-ML-SI3;2108567-79-7;ML-SI3;891016-02-7

94784696

White to off-white solid powder

1.3±0.1 g/cm3

589.3±60.0 °C at 760 mmHg

310.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.629

4

1

6

6

30

624

2

COC1=CC=CC=C1N2CCN(CC2)[C@@H]3CCCC[C@H]3NS(=O)(=O)C4=CC=CC=C4

OVTXOMMQHRIKGL-NHCUHLMSSA-N

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

N-[(1R,2R)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide

(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;

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~232.79 mM)

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.

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 (Please use freshly prepared in vivo formulations for optimal results.)