ML346 is an activator of Hsp70 expression and HSF-1 activity, with an EC₅₀ of 4.6 μM for Hsp70. ML346 restores protein folding in conformational disease models, without significant cytotoxicity or lack of specificity. ML346 induces specific increases in genes and protein effectors of the heat shock response (HSR), including chaperones such as Hsp70, Hsp40, and Hsp27^[1].

ML346 is an activator of Hsp70, with an EC₅₀ of 4600 nM in HeLa cells. ML346 (10 μM) restores proteostasis, restores CFTR-mediated iodide conductance, and enhances the correct folding of proteins expressed in two different cellular compartments^[1]. ML346 (Compound F1) induces multiple responses and strongly induces Hsp70, the oxidative stress response genes (HO1 and GCLM), and a 2.5-fold upregulation of BiP in WT MEF cells. ML346 (0.5-25 μM) exhibits cytoprotective effects in cells after a 35 min severe heat shock, and also causes a two-fold protection from H₂O₂-induced apoptosis^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

ML346 suppress the aggregation of polyQ35 in a C. elegans model, suggesting the probe has efficacy in modifying protein aggregation and associated toxicity^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

272.26

C₁₄H₁₂N₂O₄

100872-83-1

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : 12.5 mg/mL (45.91 mM; Need ultrasonic)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 1.25 mg/mL (4.59 mM); Clear solution

  此方案可获得 ≥ 1.25 mg/mL (4.59 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 12.5 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. Calamini B, et al. ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases.Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. 2012 Dec 17.

  [2]. Calamini B, et al. Small-molecule proteostasis regulators for protein conformational diseases. Nat Chem Biol. 2011 Dec 25;8(2):185-96.

In brief, HeLa cells are incubated with either DMSO (negative control), the positive controls MG132 (10 μM) and lactacystin (6 μM) or the PRs A1, A3 and ML346 (F1) for 3 and 6 hours and then harvested. Cells are lysed in homogenization buffer (50 mM Tris-HCl, pH7.5, 250 mM sucrose, 5 mM MgCl₂, 2 mM ATP, 1 mM DTT, 0.5 mM EDTA, 0.025% digitonin) for 5 min on ice, and total protein concentration of whole cell extract is determined. 3 μg of whole cell extracts are combined with assay buffer (50 mM Tris-HCl, pH 7.5, 40 mM KCl, 5 mM MgCl₂, 0.5 mM ATP, 1 mM DTT, 0.05 mg/mL BSA) in a black 96-well plate and the reaction is initiated by the addition of a 2× (200 μM) fluorogenic peptide substrate Suc-LLVY-AMC. Fluorescence is measured every 10 min using a Synergy H4 multi-mode microplate reader^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

HeLa cells are plated at a density of 10,000 cells per well in black 96-well plates in 100 μL of DMEM supplemented with 10% FBS and 1% Pen/Strep/Neo. Plates are incubated for 16 hours at 37°C, 5% CO₂ and 95% relative humidity before compound addition. 1 μL of hit compounds (ML346) in DMSO or DMSO alone are added to the sample or control wells, respectively. Plates are then placed back in the incubator for 24 hours. After incubation, cells are washed 2× with 200 μL of PBS and 200 μL of a solution of 1 μg/mL of calcein AM is added to each well. Cells are then incubated for 45 min at 37°C, 5% CO₂ before fluorescence measurement using an Analyst GT multimode reader. Percent cytotoxicity is expressed relative to wells containing cells treated with DMSO only (100%)^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Calamini B, et al. ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases.Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. 2012 Dec 17.

  [2]. Calamini B, et al. Small-molecule proteostasis regulators for protein conformational diseases. Nat Chem Biol. 2011 Dec 25;8(2):185-96.