TRPML1 (IC50 = 4.7 μM); TRPML2 (IC50 = 1.7 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]。

稳定表达hTRPML2-YFP细胞系的产生[2]
如前所述[12]，使用400mg/mL遗传霉素生成稳定表达hTRPML2-YFP细胞。如果3-4天后未发现G418抗性病灶，则将G418的浓度增加到800mg/mL。2-3周后，从G418抗性灶中取出细胞，在六孔板中扩增集落。当细胞融合率>50%时，使用共聚焦显微镜评估YFP表达。选择YFP阳性细胞超过95%的菌落，生长至>90%融合，分裂并进一步扩增

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浓度效应关系[2]
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。

HEK293细胞培养与钙显像[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。

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

70.3Ų

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)

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。