TRPML1 (IC50 = 4.7 μM); TRPML2 (IC50 = 1.7 μM); TRPML3 (IC50 = 12.5 μM)

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].

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.

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.

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 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.[1]

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The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been published, albeit without clear information about stereochemical details. In this investigation we developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer. Separation of the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 values 1.6 and 2.3 μM) and a weak inhibitor (IC50 12.5 μM) of TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC50 5.9 μM), but an activator on TRPML 2 and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first insights into structure-activity relationships in this chemotype, and showed that a broad variety of modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1 and strong activator of TRPML2).[2]

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Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of 'chatter' with ER Ca2+ channels. Our data identify new modalities for TRPML1 action.[3]

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.)