Connexin channels are proteins that form gap junctions and hemichannels in astrocytes and play a crucial role in the maintenance of the normal functions of the Central Nervous System (CNS). Alterations of astrocytic connexin expression and function in neurodegenerative diseases have been shown to affect disease progression by changing neuronal function and survival. In ALS, Cx43 gap junctions and hemichannels mediate astrocyte intercellular communication in the CNS under normal conditions and may contribute to astrocyte-mediated neurotoxicity. Targeting connexins can be a plausible therapeutic strategy to manage neurodegenerative diseases, including ALS [17, 18].
Cx43, an astrocyte protein, operates as an open pore via which toxic substances from astrocytes reach motor neurons to cause ALS. We previously performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 are transmembrane hemichannels composed of 6 subunits; they bind together to form gap junction intercellular channels. We used the program AutoDock Vina Extended for the molecular docking study. Cx31 shares amino acid and structural similarity to Cx43, and insulin docks to the same position at the N-terminal domain of monomeric Cx31 and monomeric Cx43. We found that insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS [1]. MedWatch data presented above in Table 1 confirm the protective effect of insulin.
The full Cx43 hexameric structure was deposited in the RCSB Protein Data Bank and released 8 March 2023. The results of our in silico docking study and molecular dynamics simulation confirm our previously reported findings [1]. We found that insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS
C-9 ALS is a subtype of ALS caused by a repeat expansion mutation in a gene on chromosome 9, open reading frame 72 (C9orf72). The mutation occurs when six letters of DNA – GGGGCC – are repeated hundreds of times. Besides ALS, mutations in C9orf72 can cause frontotemporal dementia. Some patients with the C9orf72 mutation develop ALS, others develop frontotemporal dementia, and some develop both. MedWatch data (Table 2) corroborate the protective effect of metformin identified in the C9orf72 ALS/FTD mouse ALS model [6].
Metformin inhibits protein kinase R, reduces RAN proteins and improves disease in C9-ALS/FTD mice. Metformin might be therapeutic for this genetic form of ALS and frontotemporal dementia because the C9orf72 mutation makes RAN proteins [6]. But metformin treatment was not effective in mice with a different form of ALS that does not produce RAN proteins [19]. Our finding that metformin docks within the Cx43 channel suggests that in some cases metformin may interfere with the passage of toxins through this channel from glial cells to motor neurons. However, because of its relatively small size compared with insulin, metformin could obstruct the Cx43 channel much less completely than insulin.
Our study has weaknesses:
A MedWatch report of an adverse event does not establish causation. For any given report, there is no certainty that the drug in question is related to the reaction. The adverse event may have been due to the underlying disease being treated, another drug being taken concurrently, or something else. The MedWatch data are imperfect, with under- and over-reporting, missing denominator (that is, number of doses for a drug), wrong, duplicate and/or missing data in the database [20]. Consequently, the total number of adverse event reports for all drugs and/or the drug in question from OpenVigil can vary slightly from drug to drug and for different adverse events related to the same drug. The imperfect MedWatch data have presented a problem that all analytical software programs, such as OpenVigil, have been forced to confront [21].
Molecular docking studies are a powerful in silico approach for discovering novel therapies for unmet medical needs by predicting drug–target interactions. But molecular docking studies are not a substitute for in vitro studies. In vitro studies are conducted in a controlled environment, such as a test tube or petri dish, and can provide more accurate results than molecular docking studies. In vitro studies can provide information about the drug's efficacy, toxicity, and pharmacokinetics, which are essential for drug development. But molecular docking studies can provide a preliminary assessment of the drug's potential efficacy and binding affinity with the target protein. Molecular docking studies are a valuable tool for drug discovery used in conjunction with in vitro studies to validate the results and ensure the safety and efficacy of the drug [22].
Molecular dynamics simulation is a computational technique that can provide mechanistic understanding of molecular systems and has become a prominent tool in pharmaceutical research. It can provide insight into the behavior of molecules at an atomic level that is difficult to characterize experimentally. However, molecular dynamics simulations are not a replacement for in vitro studies. In vitro studies are conducted in a controlled laboratory environment and can provide more accurate results than simulations. Molecular dynamics simulations can provide valuable insights into molecular systems but should be used in conjunction with in vitro studies to ensure the accuracy of the results [23].
Cx43 (7Z23) structure is presented in the closed position. Therefore, we cannot be certain of the insulin blocking effect when Cx43 is in the open position.