Identifying New AMP-Activated Protein Kinase Inhibitors That Protect against Ischemic Brain Injury

Abstract

We recently reported that AMP-activated protein kinase (AMPK) contributes to zinc-induced neuronal death by inducing Bim, a pro-apoptotic Bcl-2 homology domain 3-only protein, in a liver kinase B1 (LKB1)-dependent manner. Current data suggest AMPK plays key roles in excitotoxicity and ischemic brain injury, with zinc neurotoxicity representing at least one mechanism of ischemic neuronal death. Inhibition of AMPK could be a viable therapeutic strategy to prevent ischemic brain injury following stroke. This prompted our search for novel inhibitors of AMPK activity and zinc-induced neuronal death using cultured mouse cortex and a rat model of brain injury after middle cerebral artery occlusion (MCAO). In structure-based virtual screening, 118 compounds were predicted to bind the active site of AMPK α2, and 40 showed in vitro AMPK α2 inhibitory activity comparable to compound C (a well-known, potent AMPK inhibitor). In mouse cortical neuronal cultures, 7 of 40 compound reduced zinc-induced neuronal death at levels comparable to compound C. Ultimately, only agents 2G11 and 1H10 significantly attenuated various types of neuronal death, including oxidative stress, excitotoxicity, and apoptosis. When administered as intracerebroventricular injections prior to permanent MCAO in rats, 2G11 and 1H10 reduced brain infarct volumes, whereas compound C did not. Therefore, these novel AMPK inhibitors could be drug development candidates to treat stroke.

Mechanism of Zinc Excitotoxicity: A Focus on AMPK

“…We observed that these two chemicals noticeably attenuated ischemic brain injury in the permanent MCAO animal model. Here, we did not see any protective effect of compound C, which may be because the animal model we used experienced quite severe ischemic insults compared with those in other models (Eom et al, 2019; Figure 3). Since based on our results with compound C, the role of AMPK in cortical neuronal cultures was not related to NMDA excitotoxicity.…”

“…To find candidates with broad-spectrum efficacy against diverse cell death mechanisms in brain ischemia, we examined the protective effects of chemicals against not only zinc excitotoxicity but also calcium-overload excitotoxicity, oxidative free radical damage, and apoptosis. Two selected compounds showed substantial protective effects in a permanent MCAO model in rats (Eom et al, 2019). The success of our approach may highlight the importance of finding chemicals that can block diverse cell death mechanisms, which are likely involved in acute brain injury such as stroke.…”

“…Subsequently, after selecting 40 inhibitor substances through AMPK enzyme assay, we observed whether these 40 chemicals reduce zinc excitotoxicity comparing with compound C, a well-known chemical inhibitor for AMPK. Seven chemicals significantly inhibited zinc toxicity, but there was no discernable structural similarity (Eom et al, 2019).…”

“…Therefore, we examined seven chemicals, whether it can attenuate glutamate-or NMDA-induced excitotoxicity, H 2 O 2 -, or Fe 3+ -induced oxidative stress, staurosporine-, or etoposide-induced apoptosis. We finally chose two compounds, 2G11 and 1H10, that exhibited protective effects in all these neurotoxicity paradigms (Eom et al, 2019).…”

“…Since based on our results with compound C, the role of AMPK in cortical neuronal cultures was not related to NMDA excitotoxicity. On the other hand, the two lead compounds we selected as above have shown excellent protection in animal models, because they have suppressed not only zinc excitotoxicity, ROS-mediated oxidative stress, and apoptosis but also calcium excitotoxicity ( Eom et al, 2019 ; Figure 3 ).…”