Targeting ferroptosis for neuroprotection: Novel diphenylamine compounds targeting mitochondrial pathways of oxidative cell death
Neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease as well as stroke are of growing concern in our aging societies. In the last decades, biochemical hallmarks of these pathologies, containing excessive lipid peroxide formation, iron overload and mitochondrial impairments were li...
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|Summary:||Neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease as well as stroke are of growing concern in our aging societies. In the last decades, biochemical hallmarks of these pathologies, containing excessive lipid peroxide formation, iron overload and mitochondrial impairments were linked to recently described forms of regulated, oxidative cell death mechanisms such as oxytosis and ferroptosis. Neurons are vulnerable to mitochondrial damage due to their high energy demand associated with the high electrical activity. Thus, protecting mitochondria is a reasonable approach for preventing neuronal dysfunction and cell death. In particular, the pro-apoptotic protein BID is of central interest in the pathology of neurodegeneration. Studies using Bid KO mice showed a significant decrease in infarct volumes upon stroke induction. Moreover, Bid knock-out trials in HT22 neurons demonstrated that BID was crucially involved in the mechanisms of oxidative cell death model of oxytosis and ferroptosis, possibly participating in the transfer of the ROS-dependent upstream events to mitochondria. Hence, BID is an attractive target molecule for ferroptosis inhibition and, therefore, for the development of novel compounds targeting neurodegenerative diseases.
However, in silico compound search was always hampered by a missing high-resolution X-ray structure of the BID protein. To improve the current understanding of the protein, the previously started approaches to elucidate the 3D crystal structure of the BID protein were continued. These experiments were conducted using the established Bid3CCSS construct. Previous high-throughput screens showed that in the Morpheus A5 condition, Bid3CCSS crystals could be consistently obtained in the form of ingrown needle clusters. Due to intensive screening of the components from the Morpheus A5 condition during the presented work, crystal quality was considerably enhanced. The best crystals from this optimization process were then subjected to a streak seeding protocol and cryoprotection. X-ray measurements of the obtained non-ingrown crystals resulted in data sets for the apo Bid3CCSS crystal with a resolution of 2.0 – 2.3 Å. After successful phase determination using gadolinium-acetate-soaked crystals, a crystal structure of the BID3CCSS could be modeled for the first time. The Bid3CCSS protein crystallized as a trimer. Although the protein is constructed mainly of alpha helices, large parts of two monomers were disordered, which resulted in below-average refinement statistics. However, one monomer is well-resolved and therefore could serve as a basis for targeted in silico compound development against the BID protein in the future.
Further crystallographic experiments should be conducted to lower the protein’s flexibility in the crystal assembly, change the crystal packing by improving the crystal contacts, and identify (novel) binding pockets by the introduction of ligands.
In the second part of the thesis, novel diphenylamine (DPA) compounds were characterized in ferroptosis. These compounds were previously developed upon a medicinal chemistry approach using the first BID inhibitor BI-6c9 as a scaffold molecule to identify novel inhibitors of oxidative cell death. The selected DPA compounds 1-3 were the most potent inhibitors against glutamate induced oxytosis in preliminary experiments. Similarly, these novel DPA compounds were very potent and selective ferroptosis inhibitors. With EC50 values between 0.23-0.32 µM, the compounds were 10-20-fold more potent in preventing oxidative cell death than the previously described BID inhibitors. The DPA compounds abrogated lipid ROS formation, as the most conclusive hallmark of ferroptosis, as well as cytosolic and mitochondrial ROS formation without affecting the upstream cascade of ferroptosis including glutathione levels and GPX4 expression. Interestingly, a pronounced ROS scavenging effect by the DPA compounds could be excluded indicating that a direct antioxidant effect, possibly provided by the diphenylamine structure, was of minor relevance for the observed protective effects.
Moreover, mitochondrial respiration and constitution were maintained in a concentration-dependent manner. Post-treatment experiments substantiated the suggestion that mitochondrial protection and an interference in the ROS development processes by the compounds may contribute to the protective mechanism. Testing BID as a potential target structure in t-BID overexpression assays, failed to demonstrate a significant effect of the DPA compounds on t-BID. Moreover, preliminary binding assays of the compounds to the Bid3CCSS protein construct using MST and NMR techniques as well as co-crystallization and soaking experiments showed no clear binding activities so far, although these assays need further optimization.
In conclusion, these results highlight the role for mitochondrial protection as a strategy for the development of anti-ferroptotic compounds as possible new treatment options of neurodegenerative disorders in the future. The in vitro results encourage for further in vivo testing of the DPA compounds to evaluate the toxicity and effectiveness in models where ferroptosis might play a significant role, as for example in cerebral hemorrhage.|
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