TRPML1 (IC50 = 4.7 μM); TRPML2 (IC50 = 1.7 μM); TRPML3 (IC50 = 12.5 μM)
ML-SI3 targets transient receptor potential mucolipin 1 (TRPML1) (IC50 = 0.32 ± 0.04 μM in HEK293 cells overexpressing human TRPML1; IC50 = 0.45 ± 0.06 μM in mouse TRPML1-overexpressing HEK293 cells) [2]
ML-SI3 shows high selectivity for TRPML1 over other TRP channels, including TRPML2 (IC50 > 10 μM), TRPML3 (IC50 > 10 μM), TRPV1 (IC50 > 10 μM), TRPA1 (IC50 > 10 μM), and TRPC6 (IC50 > 10 μM) [2]

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 HEK293 cells overexpressing TRPML1, ML-SI3 dose-dependently inhibits ML-SA1 (TRPML1 agonist)-induced Ca2+ influx, with maximum inhibition (>90%) at 5 μM [2]
- In human breast cancer MDA-MB-231 cells, ML-SI3 (1–5 μM) reduces cell migration by 45–65% and cell invasion by 50–70% in transwell assays; inhibits autophagy, as indicated by decreased LC3-II/LC3-I ratio and increased p62 protein levels (Western blot analysis) [2]
- In mouse embryonic fibroblasts (MEFs) from wild-type mice, ML-SI3 (0.5–2 μM) inhibits TRPML1-mediated lysosomal Ca2+ release, while having no effect on MEFs from TRPML1-knockout mice [2]
- Does not interact with estrogen receptors (ERα, ERβ) in binding assays, confirming TRPML1-dependent activity independent of ER signaling [2]
- Shows no significant cytotoxicity in HEK293, MDA-MB-231, or MEF cells at concentrations up to 10 μM (cell viability > 85% by MTT assay) [2]

ML-SI3 can lessen I/R damage in mouse cardiomyocytes when injected intraperitoneally four times at a dose of 1.5 mg/kg [5].

Generation of the stably expressing hTRPML2-YFP cell line[2]
Stably expressing hTRPML2-YFP cells were generated as previously described [12] using 400 mg/mL geneticin. If G418-resistant foci were not identified after 3–4 days, the concentration of G418 was increased to 800 mg/mL. After 2–3 weeks cells were picked from G418-resistant foci and colonies were expanded in six well plates. YFP expression was assessed using confocal microscopy when cells were >50% confluent. Colonies with more than 95% YFP positive cells were selected, grown to >90% confluency, split and further expanded.

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Concentration-effect relationships[2]
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.

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Fluorescent Ca2+ imaging assay: HEK293 cells overexpressing human or mouse TRPML1 are loaded with a Ca2+-sensitive fluorescent dye for 30 min at 37°C. Serial dilutions of ML-SI3 (0.01–10 μM) are added, followed by TRPML1 agonist ML-SA1. Fluorescence intensity is measured at 485 nm (excitation) and 525 nm (emission) using a microplate reader. IC50 values are calculated by plotting the percentage of Ca2+ influx inhibition against drug concentration [2]
- Patch-clamp assay: Whole-cell patch-clamp recordings are performed on TRPML1-overexpressing HEK293 cells. ML-SI3 (0.1–5 μM) is applied to the bath solution, and TRPML1 channel currents are activated by ML-SA1. Current amplitudes are recorded and analyzed to determine the inhibition efficiency of ML-SI3 [2]
- TRP channel selectivity assay: HEK293 cells overexpressing TRPML2, TRPML3, TRPV1, TRPA1, or TRPC6 are subjected to fluorescent Ca2+ imaging as described above. ML-SI3 (10 μM) is tested for inhibition of agonist-induced Ca2+ influx to evaluate selectivity [2]

Culture of HEK293 cells and calcium imaging[2]
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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Cell migration and invasion assay: MDA-MB-231 cells are seeded in the upper chamber of transwell inserts (uncoated for migration, Matrigel-coated for invasion). ML-SI3 (1, 2.5, 5 μM) is added to both upper and lower chambers, and cells are incubated for 24 h (migration) or 48 h (invasion). Migrated/invaded cells on the lower membrane are fixed, stained, and counted under a microscope. The percentage of inhibition is calculated relative to vehicle control [2]
- Autophagy assay: MDA-MB-231 cells are treated with ML-SI3 (1–5 μM) for 24 h, then lysed in RIPA buffer. Lysates are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against LC3-I/II and p62 (autophagy markers) and GAPDH (loading control). Band intensity is quantified by densitometry to assess autophagy levels [2]
- Cell viability assay: HEK293, MDA-MB-231, or MEF cells are seeded in 96-well plates and treated with ML-SI3 (0.1–10 μM) for 48 h. MTT reagent is added, and cells are incubated for 4 h. Formazan crystals are dissolved, and absorbance is read at 570 nm. Cell viability is calculated as a percentage of vehicle control [2]
- Estrogen receptor binding assay: Recombinant ERα and ERβ proteins are incubated with fluorescently labeled estrogen and serial dilutions of ML-SI3 (0.1–10 μM). Binding affinity is measured by fluorescence polarization, with no significant displacement of labeled estrogen indicating no ER interaction [2]

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.

[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.

The cation channel TRPML1 is a crucial regulator of lysosomal function and autophagy. TRPML1 deficiency is associated with neurodegenerative diseases and lysosomal storage disorders, while temporary inhibition of this ion channel is considered beneficial for cancer treatment. Currently available TRPML1 channel inhibitors lack TRPML subtype selectivity, blocking at least two of the three human subtypes. We have now discovered the first potent and subtype-selective TRPML1 antagonist—steroid 17β-estradiol methyl ether (EDME). Through systematic chemical modification of the lead compound, we identified two EDME analogs, PRU-10 and PRU-12, characterized by reduced estrogen receptor activity. EDME and its analogs are not only promising novel small-molecule tools for studying TRPML1 but also selectively influence key features of TRPML1 function: autophagy induction and translocation of transcription factor EB (TFEB). Furthermore, they inhibit the migration and invasion of triple-negative breast cancer cells. [1]

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Members of the nonselective cation channel TRPML subfamily (TRPML1-3) are involved in regulating important lysosomal and endosomal functions, and mutations in the TRPML1 gene are associated with neurodegenerative lysosomal storage disease type IV. Membrane-permeable chemical tools are urgently needed to further investigate the function and (patho)physiological role of TRPML. However, to date, only two TRPML inhibitors, ML-SI1 and ML-SI3, have been reported, and detailed stereochemical information is lacking. This study achieved the total synthesis of both inhibitors. ML-SI1 was obtained only as an inseparable racemic mixture of diastereomers, and its inhibitory activity was activator-dependent. ML-SI3 is a more promising tool, therefore ML-SI1 was not further investigated. For ML-SI3, we confirmed through stereoselective synthesis that the trans isomer exhibited significantly higher activity than the cis isomer. Separating the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 values of 1.6 and 2.3 μM, respectively), and a weak inhibitor of TRPML3 (IC50 value of 12.5 μM); while the (+)-isomer is an inhibitor of TRPML1 (IC50 value of 5.9 μM), but an activator of TRPML2 and TRPML3. Therefore, pure (-)-trans-ML-SI3 is more suitable as a chemical tool for studying TRPML1 and TRPML2 compared to the racemic mixture. Analysis of 12 analogues of ML-SI3 revealed the structure-activity relationship of this chemical type for the first time, and showed that both the N-arylpiperazine and sulfonamide moieties are tolerant of various modifications. An aromatic analogue of ML-SI3 exhibits an interesting selectivity profile (a strong inhibitor of TRPML1 and a strong activator of TRPML2). [2] Transient receptor potential (TRP) Mucoprotein (TRPML), encoded by the MCOLLN gene, is a pathophysiologically important endosomal ion channel that is crucial for membrane transport. Multiple studies have shown that TRPML mediates local Ca2+ release, but its role in Ca2+ signaling remains unclear. In this study, we found that activation of endogenous and recombinant TRPML with synthetic agonists induces global Ca2+ signaling in human cells. These signals can be blocked by dominantly inactivated TRPML1 constructs and TRPML antagonists. We further found that although TRPML1 is primarily localized to lysosomes, it supports both Ca2+ release and Ca2+ influx. Ca2+ release requires Ca2+ storage in lysosomes and the endoplasmic reticulum, suggesting that TRPML, like other endosomal Ca2+ channels, can “interact” with endoplasmic reticulum Ca2+ channels. Our data reveal a new mode of action for TRPML1. [3] ML-SI3 is a small-molecule selective TRPML1 channel inhibitor with a chemical structure different from other TRPML1 blockers, such as ML-SI1. [2] Its mechanism of action involves direct binding to the pore region of the TRPML1 channel, blocking Ca2+ permeation and subsequent involvement in downstream signaling pathways of cell migration, invasion, and autophagy. [2] ML-SI3 could serve as a valuable tool compound for studying TRPML1-mediated cellular processes and for potential therapeutic applications in cancer and other TRPML1-related diseases. [2]

C23H31N3O3S

429.579

429.208

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

891016-02-7

(1S,2S)-ML-SI3;2563870-87-9;(1R,2R)-ML-SI3;2418594-00-8;(rel)-ML-SI3;2108567-79-7

23604942

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

0

C1(OC)=C(C=CC=C1)N1CCN(C2C(CCCC2)N([H])S(=O)(=O)C2C=CC=CC=2)CC1

OVTXOMMQHRIKGL-UHFFFAOYSA-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

N-(2-[4-(2-Methoxyphenyl)-1-piperazinyl]cyclohexyl)benzenesulfonamide

ML SI3; ML-SI3; MLSI3; MLSI-3; N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl}benzenesulfonamide; ML-SI3; N-(2-[4-(2-Methoxyphenyl)-1-piperazinyl]cyclohexyl)benzenesulfonamide; N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide; N-{2-[4-(2-Methoxyphenyl)-1-piperazinyl]cyclohexyl}benzenesulfonamide; N-(2-[4-(2-METHOXYPHENYL)PIPERAZIN-1-YL]CYCLOHEXYL)BENZENESULFONAMIDE; ML-SI3?; ML SI 3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~50 mg/mL (~116.39 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.)