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  • Second the phosphorylation events e

    2023-01-14

    Second, the phosphorylation events (e.g., α-Thr172 and β-Ser108) needed for AMPK activation by small molecules binding at the ADaM or other sites remains unclear. Within the β-GBD, phosphorylation of β1-Ser108 was shown to be required for the activation by small molecules binding in the ADaM site, such as salicylate, A-769662 and 991 [82], [87], [95]. In contrast to β1-Ser108, phosphorylation of β2-Ser108 is not required for AMPK activation by 991 [121], reflecting subtle changes between the β1 and β2 subunits in the topology of the β-GBD/α-KD interface in interaction with the activating ligand in the ADaM site. It is known that β1- and β2-GBD adopt different docking onto the α-KD. In addition, presence of different p-Chlorophenylalanine mg near the ADaM site between the two β-isoforms may explain why some of the small molecule activators (e.g., benzimidazole derivatives) are able to activate either β1-containing or both β1- and β2-containing AMPK isoforms [78]. Consistently, generation of chimeras in which nonconserved amino acids proximal to this interface have been swapped from β1 to β2 leads to altered ligand specificity [81]. Crystal structure of a β2-containing isoform that contains a bound ligand at the ADaM site will help to better understand the molecular basis of these differences and may bring novel knowledge on the pharmacological regulation of AMPK. This information might be useful to design novel isoform or tissue-selective compounds minimizing unwanted side effects. Third, it was originally reported that β1-Ser108 is an autophosphorylation site [122], but it was recently reported as a substrate for ULK1 under conditions associated with elevated AMP [47]. In an aspect relevant to therapeutics, these findings are significant given the importance of this post-translational modification in the mechanism of action of most AMPK synthetic activators and could impact on AMPK drug sensitization independently of autophosphorylation. Interestingly, ULK1 phosphorylation of Ser108 was shown to be specific to the AMPK β1-isoform due to divergence in the sequence surrounding Ser108 in the β2-isoform [47]. It is not known whether other kinases can phosphorylate β2-Ser108. Fourth, an exception for the requirement of both α-Thr172 and β-Ser108 phosphorylation is synergistic activation of “naive” unphosphorylated AMPK with compounds binding simultaneously at the ADaM site and γ subunit (e.g., A-769662 or 991 and AMP, respectively) as shown in cell-free essays [121], [122], [123]. Binding of small molecule in the ADaM site appears to be sufficient to stabilize the activation loop in an active conformation [121]. Interestingly, combination of A-769662 with C2 also resulted in a synergistic allosteric activation of AMPK bypassing the need for AMPK-α Thr172 phosphorylation [85]. These findings have important implications for development of AMPK-targeting therapeutics and point to possible combinatorial therapies based on synergistic activation of AMPK by direct small molecule allosteric activators independent of AMP and in p-Chlorophenylalanine mg the absence of upstream phosphorylation (e.g., due to genetic loss or downregulation of LKB1 signaling [96], [124]). Discovery of a mechanism that induces AMPK drug sensitization independently of autophosphorylation also provides a potential strategy to treat non-small-cell lung and cervical carcinomas, associated with genetic loss of LKB1. However, whether phosphorylation of Thr172 is absolutely required for AMPK signaling in intact cells remains controversial. While Dite et al. reported that combination of A-769662 and phenformin can trigger AMPK cellular signaling independently of α-Thr172 phosphorylation [47], Willows et al. failed to activate AMPK signaling in the presence of 991 and 2-deoxyglucose in cells lacking both LKB1 and CaMKK2 [121]. Fifth, while small molecule AMPK activators hold promise for the treatment of a number of human pathologies, concerns about adverse effects of systemic and chronic AMPK activation were raised by studies from naturally occurring single point mutations within the human PRKAG2 gene [125], [126]. These mutations have been associated with increased basal activity of AMPK and are associated with deleterious metabolic cardiac phenotype and hypertrophy. In addition, a genetic mouse model with analogous mutation showed hyperphagia, obesity and impaired β-cell function, questioning the safety of chronic and generalized AMPK activators [118]. Henceforth, establishing efficacy and safety would be essential before AMPK activators would be appropriate for introduction into clinical studies. The potential cardiac safety concerns associated with chronic systemic AMPK activation with MK-8722 have been recently evaluated in rhesus monkeys dosed for up to 8 months [111]. Although increased cardiac glycogen content and hypertrophy was observed but without any changes in electrocardiogram and apparent functional cardiac sequelae, any safety issues associated with AMPK activators remains to be carefully determined in phase I trials. However, the exciting possibility of safe AMPK-driven therapeutics is encouraged by the recently reported advancement and successful completion of phase I trials for two novel small molecule AMPK activators, PXL770 and O304, from Poxel SA and Betagenon AB, respectively [127], [128].