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ItemMechanisms of activation and inhibition of the metabolic regulator AMP-activated protein kinase (AMPK)Dite, Toby ( 2018)Biochemical energy in the form of ATP is crucial for normal metabolic function of all cells. Relative levels of AMP, ADP and ATP reflect the energy status of the cell, and are described in a single parameter called the adenylate energy charge. The master metabolic regulator of the cell, AMP-activated protein kinase (AMPK), maintains energy homeostasis by responding to fluctuations in adenylate energy charge, and adjusting ATP consuming and producing pathways. AMPK activation can lower plasma glucose, reduce de novo cholesterol and fatty acid synthesis and increase fatty acid oxidation, which has encouraged the development of many small molecule activators that directly activate AMPK. AMPK activity can also play a deleterious role in cancer cell survival, neurodegeneration and stroke, which has alternatively highlighted AMPK inhibition as a potential therapeutic strategy, although small molecule antagonists are currently limited. Contextualising AMPK regulation in an intracellular environment requires simultaneous monitoring of adenine nucleotide ratios. While this is not routine in the field, I have used mass spectrometry to highlight several scenarios where quantitation of adenine nucleotide ratios is critical. I have shown that glucose starvation is able to decrease adenylate energy charge in some cells, but not others. I have also shown that high concentrations of any small molecule may indirectly activate AMPK by increasing [AMP]/[ATP]. Finally, whereas the adenylate kinase equilibrium predicts that [AMP]/[ATP] will increase as the square of [ADP]/[ATP] during stress, I have shown empirically that, under most circumstances, this is not the case. Together, these results demonstrate that measuring adenine nucleotide ratios is fundamental to distinguish novel mechanisms from canonical mechanisms of intracellular AMPK regulation. I next investigated the regulation of AMPK by allosteric drugs. The allosteric drug and metabolite (ADaM) site on AMPK is the most commonly reported drug binding site in AMPK/drug crystal structures. Work conducted prior to my PhD has shown that phosphorylation of AMPK at β1-Ser108 stabilises the ADaM site and is critical for activation by many drugs, and enhances activation by others. I have identified and confirmed ULK1 as an upstream kinase of AMPK β1-Ser108. Phosphorylation of AMPK by ULK1 sensitized AMPK to activation by two allosteric drugs, A-769662 and salicylate. Pharmacological inhibition or genetic deletion of ULK1 in cells reduced β1-Ser108 phosphorylation on recombinant AMPK in cells, and increases in β1-Ser108 phosphorylation. In addition, mutation of Thr172 to Ala did not prevent AMPK pathway signalling under circumstances of increased β1-Ser108 phosphorylation in the presence of A-769662, demonstrating the first activation loop-independent AMPK signalling in cells. Finally, I have identified and characterised SBI-0206965 as a novel type II inhibitor of AMPK. SBI-0206965 was able to inhibit both α1- and α2-AMPK heterotrimers with nanomolar IC50 values. SBI-02069965 displayed preferable potency and selectivity profiles compared to compound C, and was able to inhibit cellular AMPK signalling across a range of AMPK-activating stimuli. Co-crystallisation of AMPK α2-kinase domain with SBI-0206965 revealed that it binds to AMPK at the active site, in a catalytically inactive, DFG-out conformation. Biochemical characterisation of SBI-0206965 showed that, with regard to ATP competition, it is a mixed-type inhibitor, and is able to maintain potency in activity assays when [ATP] is high. This discovery is the first example of a type II AMPK inhibitor, which is more selective and potent that currently available inhibitors. SBI-0206965 is a promising lead for small molecule AMPK inhibitor development against a backdrop of increasing applications for AMPK inhibition. In conclusion, work in this thesis provides insights into mechanisms of AMPK activation and inhibition that may be useful for the development of AMPK drugs with clinical potential. Current small molecule activators of AMPK have not reached clinical trials, and the handful of existing small molecule inhibitors of AMPK are poor. Exploiting endogenous mechanisms that increase AMPK β1-Ser108 phosphorylation, such as activating ULK1, could be used as a potential strategy for increasing AMPK pathway activation by drugs. Using SBI-0206965 as a research tool, and as a platform for the development of even better antagonists will further enhance our understanding of the roles of AMPK, and may lay the foundation for a new class of therapeutic AMPK inhibitors. Essential to the understanding of both AMPK activation and inhibition will be the adoption of adenine nucleotide quantitation as standard practice in the field.