Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite-binding site.

AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.

Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965.

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 µM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.

Blocking AMPK ß1 myristoylation enhances AMPK activity and protects mice from high-fat diet-induced obesity and hepatic steatosis.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK ß subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch" mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKß1 (ß1-G2A). We demonstrate that non-myristoylated AMPKß1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of ß1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the ß1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.

Targeted Protein Degradation through Cytosolic Delivery of Monobody Binders Using Bacterial Toxins.

Monobodies are small engineered binding proteins that, upon expression in cells, can inhibit signaling of cytosolic oncoproteins with outstanding selectivity. Efficacy may be further increased by inducing degradation of monobody targets through fusion to the von Hippel-Lindau (VHL) substrate receptor of the Cullin2-E3 ubiquitin ligase complex. However, potential therapeutic use is currently limited, because of the inability of monobody proteins to cross cellular membranes. Here, we use a chimeric bacterial toxin, composed of the Shiga-like toxin B (Stx2B) subunit and the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA-II) for delivery of VHL-monobody protein fusions to target endogenous tyrosine kinases in cancer cells. Depending on the expression of the Stx2B receptor Gb3 on the cell surface, we show that monobodies are taken up by an endocytic route, but are not degraded in lysosomes. Delivery of monobodies fused to a nuclear localization signal resulted in accumulation in the nucleus, thereby indirectly, but unequivocally, demonstrating cytosolic delivery. Delivery of VHL fused to monobodies targeting the Lck tyrosine kinase in T-cells resulted in reduced Lck protein levels, which was dependent on the expression of Gb3. This led to the inhibition of proximal signaling events downstream of the T-cell receptor complex. This work provides a prime example of the delivery of a stoichiometric protein inhibitor of an endogenous target protein to cells and inducing its degradation without the need of genetic manipulation of target cells. It lays the foundation for further in vivo exploitation of this delivery system.