Background
IC50: 2.9 nM and 1.1 nM for ULK1 and ULK 2, respectively.
MRT68921 is a dual autophagy kinase ULK1/2 inhibitor.
Autophagy is found to be a critical cell-degradative and protective process recycling damaged and long-lived cellular components. ULK1 is a serine/threonine protein kinase that is important for the initial stages of autophagy.
In vitro: MRT68921 was identified to be relatively specific, but still could inhibit a number of kinases by over 80%. Most importantly, TBK1/IKK as well as the AMPK-related kinases were still found to be targeted. Moreover, by using LKB1 knock-out MEFs, it was found that LC3 flux was comparable with matched, wild-type MEFs and could be inhibited to the same extent in the treatment with MRT68921. Thus, these kinases are suggested to be not the target of MRT68921 in blocking autophagy. In addition, MRT68921 was found to be able to block the WT-restored ATG13 phosphorylation and autophagy, which was similar to cells expressing endogenous ULK1. In contrast, in cells expressing a similar level of M92T ULK1, MRT68921 could not reduce either ATG13 phosphorylation or LC3 flux, which indicated that MRT68921 was truly able to block autophagy via ULK1 kinase inhibition [1].
In vivo: So far, there is no animal in vivo data reported.
Clinical trial: Up to now, MRT68921 is still in the preclinical development stage.
Reference:
[1] Petherick KJ, Conway OJ,Mpamhanga C,Osborne SA,Kamal A,Saxty B,Ganley IG.? Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy. J Biol Chem.2015 May 1;290(18):11376-83.
Protocol
Kinase experiment: | Kinase assays are carried out in 50 mM Tris-HCl, pH 7.4, 10 mM magnesium acetate, 0.1 mM EGTA, and 0.1% β-mercaptoethanol, containing 30 μM cold ATP, and 0.5 μCi of [γ-32P]ATP for 5 min at 25 °C. Prior to ATP addition, reaction mixes are pre-warmed to 25 °C for 5 min. Reactions are stopped by the addition of sample buffer, followed by SDS-PAGE, transfer to nitrocellulose, and analysis by autoradiography and immunoblot[1]. |
Cell experiment: | MEFs and 293T cells are grown in DMEM, supplemented with 10% fetal bovine serum and penicillin/streptomycin, and cultured at 37°C, 5% CO2. For induction of autophagy, cells are typically grown to 75% confluency, ished twice, and incubated in Earle's balanced salt solution (EBSS) for 1 h (or complete medium as a control). MRT67307 (10 μM), MRT68921 (1 μM), AZD8055 (1 μM), or bafilomycin A1 (50 nM) is included[1]. |
参考文献: [1]. Petherick KJ, et al. Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy. J Biol Chem. 2015 May 1;290(18):11376-83. |
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