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

Sanders, Matthew J.; Ratinaud, Yann; Neopane, Katyayanee; Bonhoure, Nicolas; Day, Emily; Ciclet, Olivier; Lassueur, Steve; Pinta, Martine Naranjo; Deák, Mária; Brinon, Benjamin; Christan, Stefan; Steinberg, Gregory R.; Barron, Denís; Sakamoto, Kei

doi:10.1016/j.jbc.2022.101852

Cited by 8 publications

(12 citation statements)

References 62 publications

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“…Possible reasons are numerous and include the uncharacterised influence of affinity tags used to purify AMPK, substrate composition, assay conditions, kinase detection method and source of recombinant AMPK that influences regulatory post-translational modifications. In terms of the latter, AMPK activation by extended CaMKK2 treatment, common practice in high throughput screening platforms [17,18,35], generates preparations with supraphysiological autophosphorylation of β-Ser108 (>95% vs. <10% basal β-pSer108 stoichiometry from mammalian cells [36,37]). Phosphorylation of β1-Ser108 stabilises the ADaM site and is required for AMPK sensitisation to lower potency activators A-769662, MT47-100, salicylate, long-chain fatty acyl CoA esters and lusianthridin [12,15,16,20,35,37], where the loss of phosphorylation via exchange for Ala reduces activating potency of SC4 by ∼4-fold and 991 by 40-fold [20,21].…”

Section: Discussionmentioning

confidence: 99%

“…In terms of the latter, AMPK activation by extended CaMKK2 treatment, common practice in high throughput screening platforms [17,18,35], generates preparations with supraphysiological autophosphorylation of β-Ser108 (>95% vs. <10% basal β-pSer108 stoichiometry from mammalian cells [36,37]). Phosphorylation of β1-Ser108 stabilises the ADaM site and is required for AMPK sensitisation to lower potency activators A-769662, MT47-100, salicylate, long-chain fatty acyl CoA esters and lusianthridin [12,15,16,20,35,37], where the loss of phosphorylation via exchange for Ala reduces activating potency of SC4 by ∼4-fold and 991 by 40-fold [20,21]. To our knowledge the influence of β1-S108 phosphorylation on AMPK activation kinetics by MK-8722 and PF-739 has not been reported, however a similar sensitising effect is likely for all high potency pan activators and consequently assays using highly activated material may be expected to output more potent kinetics than those using more physiologically relevant preparations.…”

Section: Discussionmentioning

confidence: 99%

“…Increasing evidence indicates that ADaM site-targeting activators may induce energy stress at high concentrations, leading to phosphorylation of α-Thr172 by canonical AMP-dependent mechanisms (most recently investigated by Sanders and colleagues [35]) and supporting our previous recommendation that α-pThr172 should not be used in isolation as a marker of AMPK drug bioactivity [37]. Elevated cellular α-pThr172 is often used to demonstrate on-target action of AMPK drugs and is explained mechanistically by a drug-induced conformational change that makes α-pThr172 resistant to dephosphorylation.…”

Section: Discussionmentioning

confidence: 99%

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Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

Ovens

Gee

Ling

et al. 2022

Biochemical Journal

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The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and a2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

Ovens

Gee

Ling

et al. 2022

Biochemical Journal

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“…Total and phosphorylated AMPK levels were measured using specific primary antibodies (Cell Signaling #2531 (Koh et al, 2022); Cell Signaling #2535 (Bartolacci et al, 2022)-recognizes phosphorylated Threonine 172). Total raptor (Cell Signaling #2280 (Sanders et al, 2022)) and phosphorylated raptor (S792, Cell Signaling #2083 (Sanders et al, 2022)) levels were also measured alongside total P70S6K (Cell Signaling #9202 (Zhang et al, 2021)), phosphorylated P70S6K (Santa Cruz SC-11759 (Xiao et al, 2002)), total ULK (Cell signaling #8054 (Wu et al, 2020)), and ULK S555 (cell signaling #5869 (Wu et al, 2020)). Synaptophysin (Cell Signaling #5461 (Wu et al, 2018)), NeuN (Cell Signaling #24307 (Tang et al, 2021)), PSD-95 (post-synaptic marker, Cell Signaling #36233 (Shui et al, 2022)), Homer-1 (post-synaptic marker, SC-136358 (Wang et al, 2014) (Tang et al, 2019)), mTOR S2448 (Cell signaling #2971 (Luo et al, 2022)), total mTOR (Cell signaling #2972 (Luo et al, 2022)), Akt T308 (Cell signaling #9275 (Mathieu et al, 2019)), Akt S473 (Cell signaling #4058 (Marko et al, 2020)), and total Akt (Cell signaling #4685 (Marko et al, 2020)) were measured as markers of insulin signaling.…”

Section: Western Blottingmentioning

confidence: 99%

Influence of metabolic stress and metformin on synaptic protein profile in SH‐SY5Y‐derived neurons

Yang,

Mohammad,

Finch

et al. 2023

Physiological Reports

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Insulin resistance (IR) is associated with reductions in neuronal proteins often observed with Alzheimer's disease (AD), however, the mechanisms through which IR promotes neurodegeneration/AD pathogenesis are poorly understood. Metformin (MET), a potent activator of the metabolic regulator AMPK is used to treat IR but its effectiveness for AD is unclear. We have previously shown that chronic AMPK activation impairs neurite growth and protein synthesis in SH‐SY5Y neurons, however, AMPK activation in IR was not explored. Therefore, we examined the effects of MET‐driven AMPK activation with and without IR. Retinoic acid‐differentiated SH‐SY5Y neurons were treated with: (1) Ctl: 24 h vehicle followed by 24 h Vehicle; (2) HI: 100 nM insulin (24 h HI followed by 24 h HI); or (3) MET: 24 h vehicle followed by 24 h 2 mM metformin; (4) HI/MET: 24 h 100 nM insulin followed by 24 h 100 nM INS+2 mM MET. INS and INS/MET groups saw impairments in markers of insulin signaling (Akt S473, mTOR S2448, p70s6k T389, and IRS‐1S636) demonstrating IR was not recovered with MET treatment. All treatment groups showed reductions in neuronal markers (post‐synaptic marker HOMER1 mRNA content and synapse marker synaptophysin protein content). INS and MET treatments showed a reduction in the content of the mature neuronal marker NeuN that was prevented by INS/MET. Similarly, increases in cell size/area, neurite length/area observed with INS and MET, were prevented with INS/MET. These findings indicate that IR and MET impair neuronal markers through distinct pathways and suggest that MET is ineffective in treating IR‐driven impairments in neurons.

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“…In addition to acting as a structural scaffold, the β subunit is also (auto)phosphorylated and myristoylated to allosterically regulate enzymatic activity ( 9 – 13 ). Of these posttranslational modifications, phosphorylation of AMPKβ1 Ser108 has been shown to play a vital role in enhancing AMPK activity in response to many distinct, small-molecule, allosteric activators including A769662, salicylate, 991, MK-8722, PF-06409577, and lusianthridin ( 14 – 20 ) (reviewed in ref. 6 ).…”

mentioning

confidence: 99%

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

Desjardins

Smith

Day

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

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

Cited by 8 publications

References 62 publications

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

Influence of metabolic stress and metformin on synaptic protein profile in SH‐SY5Y‐derived neurons

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

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