FMK 9a is an autophagin-1 inhibitor with IC₅₀ values of 80 and 73 μM in FRET and LRA assay.

IC50: 80 nM (autophagin-1, FRET assay); 73 nM (autophagin-1, LRA assay)^[1]

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. FMK 9a shows strong inhibition of ATG4B, with IC₅₀ values of 80 and 73 nM in the TR-FRET and cellular-based LRA assays, respectively. LC−MS/MS study indicates that FMK 9a forms an irreversible covalent bond with the more reactive thiol group of Cys74 located at the catalytic site of ATG4B and thus inactivates ATG4B proteolytic activity^[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.

FMK 9a shows moderate solubility (LYSA: 41 μg/mL) and high human and mouse liver microsome clearances of 13.9 and 70 mL/kg per minute^[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.

392.42

C₂₃H₂₁FN₂O₃

1955550-51-2

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 : 100 mg/mL (254.83 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    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (6.37 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.37 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

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

  Solubility: ≥ 2.5 mg/mL (6.37 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.37 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 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
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (6.37 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.37 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Qiu Z, et al. Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors. ACS Med Chem Lett. 2016 Jun 25;7(8):802-6.

FMK 9a is dissolved in DMSO. 5 μL of serial-diluted FMK 9a (1% DMSO) in assay buffer is added into 5 μL of purified ATG4B (final 0.1 nM) in 384-well plate, and the solution is incubated at room temperature (rt) for 30 min. Then, 5 μL of His-GATE-16-GST (final 20 nM) is added into the wells, and the solution is incubated for another 30 min. 5 μL of detection solution (final 2 nM of Eu-anti-His and 20 nM of Ulight-anti-GST) is added, and the resulting mixture is incubated at rt for 40 min^[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.

• [1]. Qiu Z, et al. Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors. ACS Med Chem Lett. 2016 Jun 25;7(8):802-6.