TRPML1 (IC50 = 1.6 μM); TRPML2 (IC50 = 2.3 μM); TRPML3 (IC50 = 12.5 μM)

HeLa 细胞的 ML-SA1 诱导的 Ca2+ 信号传导被 ML-SI3 (10 μM) 抑制 [2]。 ML-SI3（25-75 μM，24 小时）会破坏成虫血吸虫膜的完整性 [3]。在模拟的溶酶体腔中，雷帕霉素诱导的 ITRPML1 被 ML-SI3 (10 μM) 阻断 [4]。在新生大鼠心室肌细胞 (NRVM) 中，ML-SI3（3 µM，6 小时）完全消除缺氧/复氧 (H/R)（4 小时 H/2 小时 R）引起的 LC3II 和 p62 水平升高[ 5]。

当以 1.5 mg/kg 的剂量腹腔注射四次时，ML-SI3 可以减轻小鼠心肌细胞的 I/R 损伤 [5]。

浓度效应测量基于Fluo-4/AM测定，并使用内置于机器人液体处理站的定制荧光成像板阅读器（FLIPR）进行。所有成像实验均在HEPES缓冲溶液（HBS）中进行，该溶液含有132 mM NaCl、6 mM KCl、1 mM MgCl2、1 mM CaCl2、5.5 mM d-葡萄糖、10 mM HEPES，pH 7.4。溶解在DMSO（10 mM）中的化合物在HBS（0.98μM-1 mM）中连续预稀释。将稳定表达靶向人TRPML1、TRPML2或TRPML3[14]的质膜的HEK293细胞胰蛋白酶化，并重新悬浮在补充有4μM Fluo-4/AM的细胞培养基中。在37°C下孵育30分钟后，对细胞悬浮液进行短暂离心，重新悬浮在HBS中，并分配到黑色着色、底部透明的384孔微孔板中。然后将平板放入FLIPR中，用Zyla 5.5相机和μManager软件记录荧光信号（激发470nm，发射515nm），如前所述。在第一步和视频中，帝肯96尖端多通道臂向细胞中添加了阴性HBS对照或预稀释化合物，最终浓度为0.098μM–100μM。为了绘制拮抗作用，随后在每个孔中吸取ML-SA1（5μM），并记录荧光信号10分钟。通过ImageJ计算每个孔和背景区域的荧光强度进行分析。最后，减去背景，将荧光强度归一化为初始强度（F/F0）。为了比较化合物的抑制效力，对阴性对照进行了第二次归一化。将所有浓度-效应曲线拟合为四参数Hill方程，以获得Imin、Imax、IC50）和Hill系数n。[2]

如前所述，使用Fura-2进行单细胞Ca2+成像实验。将稳定表达hTRPML1ΔNC-YFP、hTRPML2 YFP或hTPPML3-YFP的HEK293细胞在37°C、5%CO2的Dulbecco改良Eagle培养基中培养，补充10%胎牛血清、100 U/mL青霉素和0.1 mg/mL链霉素。将细胞铺在涂有聚赖氨酸（sigma）的玻璃盖玻片上，并生长2-3天。对于Ca2+成像实验，细胞在37°C下用Fura-2 AM（4.0μM）和0.005%（v/v）pluronic酸在HEPES缓冲溶液（HBS）中加载45分钟，该溶液包含138 mM NaCl、6 mM KCl、2 mM MgCl2、2 mM CaCl2、10 mM HEPES和5.5 mM d-葡萄糖（用NaOH调节pH至7.4）。装载后，用HBS洗涤细胞并将其安装在成像室中。实验如前所述进行。用激活剂（10μM）刺激200秒后，再施加抑制剂（10μM）200秒。激活标准化为1。所有记录均在Leica DMi8活细胞显微镜或Polychrome IV单色仪上在HBS中进行（仅用于瞬时转染hTRPML1 HEK293细胞的实验）。Fura-2在340nm/380nm处被激发。使用515nm长通滤光片捕获发射的荧光。化合物在DMSO中预稀释，并在-20°C下作为10 mM储备溶液储存，不超过三个月。工作溶液在使用前直接用HBS稀释制备。在所有Ca2+成像实验的统计分析中，至少三个独立实验的平均值如所示。***表示p<0.001，**表示p<0.01，**表示p<0.05，ns=不显著，单因素方差分析检验，然后进行Tukey事后检验。[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.

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]

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

70.3Ų

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

注意： 本产品在运输和储存过程中需避光。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.82 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。