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A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance

Abstract

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

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Fig. 1: pAmpkα undergoes SCFFbxo48-mediated ubiquitin proteasomal degradation.
Fig. 2: Small-molecule Fbxo48 inhibitors increase pAmpkα.
Fig. 3: Fbxo48 inhibitor interrupts Fbxo48/pAmpkα interaction.
Fig. 4: BC1618 facilitates mitochondrial fission and autophagy.
Fig. 5: BC1618 improves hepatic insulin sensitivity in diet-induced obese mice.

Data availability

All relevant data are available from the authors and/or included in the manuscript or Supplementary Information. Source data are provided with this paper. Other data that support the findings of this study are available from the corresponding author upon reasonable request. Phospho-proteomics data can be accessed at MassIVE database under ID MSV000086334.

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Acknowledgements

This work was supported in part by grant no. 16SDG27650008 from the American Heart Association to Y.L. and a US National Institute of Diabetes and Digestive and Kidney Diseases grant to Y.L. and M.J.J. (1R01DK119627). This work was also supported by NHLBI grants nos. UH3HL123502, R01HL096376, R01HL097376, R01HL098174, R01HL081784 and P01HL114453 to R.K.M.; T32HL110849 to T.B.L.; 5R01HL142777 to Y.L.; 5R35HL139860 and 5R01HL133184 to B.B.C. This work was also supported in part by the United States Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, a Merit Review Award from the United States Department of Veterans Affairs to R.K.M.

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Authors

Contributions

Y.L., M.J.J. and B.B.C. designed the study, performed experiments, analyzed results and wrote the manuscript. T.B.L., B.L., M.B.L., J.R.K., M.K.N., F.T., Y.C. and B.R.H. performed experiments and analyzed data. Y.J. designed experiments. S.P.M. directed the NASH human study. C.P.O. directed animal studies. C.P.O., T.F. and R.K.M. edited the manuscript. Y.L., R.K.M. and B.B.C. directed the study.

Corresponding authors

Correspondence to Yuan Liu or Bill B. Chen or Rama K. Mallampalli.

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Competing interests

A provisional patent application (US patent 62/404,592) has been converted to a Patent Cooperation Treaty application jointly by the US Department of Veterans Affairs and the University of Pittsburgh covering all chemical entities included in this study. Y.L., T.F. and B.B.C. are the founders and employees of Generian Pharmaceuticals and may own company stock.

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Extended data

Extended Data Fig. 1 pAmpkα undergoes ubiquitin proteasomal degradation.

a, BEAS-2B cells were pretreated in glucose free media with increasing concentrations of metformin for 4h. Whole cell extracts were subjected to TUBE agarose beads pull downs (PD) and immunoblotting (Results are representative of three independent experiments). b, Fed and overnight starved mouse liver extracts were subjected to TUBE agarose beads pull down (PD) and immunoblotting (Results are representative of three independent experiments). c, BEAS-2B cells were transfected with either WT or T172A mutant Ampk. Whole cell extracts were subjected to TUBE agarose beads pull down (PD) and immunoblotting. Arrows indicate polyubiquitylated products in each blot (Results are representative of three independent experiments). Source data

Extended Data Fig. 2 pAmpkα ubiquitylation and degradation is mediated by an F-box protein.

a, BEAS-2B cells were incubated in glucose free 2% DMEM media containing DMSO, MG132 (20 µM, MG) or MLN4924 (5 µM) for 1.5 h. Cells were then collected for immunoblotting (Results are representative of two independent experiments). b, BEAS-2B cells were preincubated in glucose free 2% DMEM media containing DMSO, MG132 (20 µM) or MLN4924 (5 µM) for 1 h. CHX (40 µg/ml) was then added at indicated time points. Cells were collected at same time for immunoblotting (Results are representative of two independent experiments). c, BEAS-2B cells were incubated in high glucose then switched to glucose free 2% DMEM media for 16 h. Total RNA were purified using Qiagen RNeasy Plus Mini Kit. RNA sequencing was then performed by the Quick Biology commercial service. Differentially expressed genes of F-box protein families were plotted and 30% expression level difference was indicated in the red line cut off (Data are the mean of two biological replicates). Source data

Extended Data Fig. 3 Screening of F-box proteins involved in pAmpkα degradation.

a, BEAS-2B cells were nucleofected with a series of V5 tagged F-box protein encoding plasmids (3 µg). After incubation for 48 h, high glucose media was switched to glucose free 2% FBS DMEM media for 1 h. Cells were then collected for immunoblotting (Results are representative of two independent experiments). b, c, BEAS-2B cells were nucleofected with increasing amounts of either Skp2-V5 or Fbxl5-V5 plasmids. After incubation for 48 h, growth media were changed to glucose free media for 1 h before immunoblotting (Results are representative of two independent experiments). NS indicates a non-specific band. d,. In vitro ubiquitinylation of Ampk. Recombinant ubiquitin E1, E2 conjugating enzymes and ubiquitin was incubated with or without Fbxo48 with pAmpk and polyubiquitinylated products were detected on immunoblots (arrows) (Results are representative of two independent experiments). Source data

Extended Data Fig. 4 BC1618 off-targeting kinase screen.

The inhibitory activities for BC1618 (10 µM) against 51 kinases (ABL, CSK, EGFR, EPHA2, EPHB4, FGFR1, FLT3, IGF1R, ITK, JAK3, KDR, LCK, MET, PDGFRα, PYK2, SRC, SYK, TIE2, TRKA, TYRO3, AKT1, AMPKα1/β1/γ1, AMPKα2/β1/γ1, AurA, CaMK4, CDK2/CycA2, CHK1, CK1ε, DAPK1, DYRK1B, Erk2, GSK3β, HGK, IKKβ, IRAK4, JNK2, MAPKAPK2, MST1, NEK2, p38α, p70S6K, PAK2, PBK, PDK1, PIM1, PKACα, PKCα, PKD2, ROCK1, SGK, TSSK1) were measured and percentage inhibition of kinase activity was plotted. 50% change of kinase activities is indicated by a red line cut off line (CarnaBio) (Data are the mean of two biological replicates). Source data

Extended Data Fig. 5 PTM-SEA analysis of BC1618 phosphoproteome.

a, BEAS-2B cells were treated with BC1618 (3µM) or vehicle control and the alterations in the phosphoproteome were assessed using mass spectrometry. Heatmap of those phosphosites with p<0.05 (t-test) comparing BC1618 (left side) to DMSO control (right). Color indicates log2 normalized intensity values as row z-score. Row names indicate the corresponding protein and phosphorylation site. b, Volcano plot of statistical significance (-log10 p. value, y-axis) and the effect size (log2 scaled fold-change, x-axis) of BC1618 treated cells compared to DMSO control, where positive values represent phosphosites higher in BC1618 treated cells. c, Volcano plot of enrichment of various phosphoproteome signatures. The x axis indicates the normalized enrichment score (NES) between BC1618 and DMSO control, with positive values indicating shared phosphosite signatures and negative values indicating opposed phosphosite signatures. Dot size indicates the percent overlap of phosphosites associated with the signature and experimentally assayed phosphosites. Red indicates significantly upregulated signatures while green indicates significantly downregulated signatures (permutation-based FDR < 0.05). Grey indicates non-significant signatures (Data are from three biological replicates). Source data

Extended Data Fig. 6 BC1618 in vivo toxicity studies.

(a-d) C57BL/6 mice were given BC1618 in drinking water at 15 mg/kg/d (low) and 30 mg/kg/d (high) doses for 3 months. Mice were then euthanized, and plasma samples were collected and assayed for markers of cytotoxicity. Alanine aminotransferase (ALT) activity, creatine kinase activity, lactate dehydrogenase (LDH) content, and creatinine content were measured following the manufacturer’s protocol. e, Major organs were collected and processed for H&E staining; scale bar: 100 μm. Data represented mean +/- SEM. Data points represent n=7–8 independent mice per group, and p-values are indicated, as calculated by one-way ANOVA with Dunnett’s test of multiple comparisons (A-D). Source data

Extended Data Fig. 7 BC1618 is anti-inflammatory in vitro.

a, THP1 cells stably expressing an NF-κB inducible luciferase (secreted) reporter were treated with LPS (100 ng/ml) and BC1618 at indicated concentrations for 6 h or 24 h before supernatant was collected for luciferase activity assay following the manufacturer’s protocol. Data are shown as the mean ± SEM of three independent biological replicates, and significance was measured by one-way ANOVA with Dunnett’s test of multiple comparisons relative to 0µM BC1618+LPS. For 6hs, 0.0032µM: p=0.0868; for 0.016,0.08, 0.4, 2, and 10µM: p<0.0001. For 24hr, 0.0032µM: p=0.0002; for 0.016,0.08, 0.4, 2, and 10µM: p<0.0001. b, 50K PBMC cells were cultured in 96 well plates before being exposed to BC1618 at indicated concentrations for 18 h. Cells were then treated with LPS (10 ng/ml) for an additional 4 h. Media were then collected and TNF and IL-1 concentration were determined by ELISA. Data are shown as the mean ± SEM of three independent biological replicates, and significance was measured by one-way ANOVA with Dunnett’s test of multiple comparisons relative to 0µM BC1618 for both cytokines. For TNF treatment, 0.008µM: p=0.2212; 0.04µM: p=0.0375; 0.2µM: p=0.3585; for 1, 5, and 25µM: p<0.0001. For IL-1 treatment, 0.008µM: p=0.7497; 0.04µM: p=0.0919; 0.2µM: p=0.0126; 1µM: p=0.0028; 5µM: p=0.0015; 25µM: p=0.0065. Significance is also indicated as follows: *, P <0.05; **, P < 0.01; ***, P <0.001; ****, P<0.0001 (c) PBMC cells (1 mL at 1.0 x 106/ml) were treated with BC1618 (5 µM) for 18 h. Cells were then treated with 100 ng/ml LPS for additional 4 h. Cytokine release was monitored by the human cytokine array (R&D systems). The results from a cytokine array dot blot are shown and quantitated (Data are shown as the mean of two independent biological replicates). Source data

Extended Data Fig. 8 BC1618 reduces lung inflammation in endotoxin treated mice.

C57BL/6 mice were administered i.p. with vehicle, 2 or 10 mg/kg of BC1618. Mice were then immediately challenged with LPS (3 mg/kg) for an additional 18 h. Mice were euthanized and lungs were lavaged with saline, harvested, and then homogenized. Bronchoalveolar lavage (BAL) protein (a), cell counts (b) and cytokines (c-e) were measured. f, Representative mouse lung tissue H&E staining; scale bar: 100 μm. Data represented mean +/- SEM (n=5 independent mice per group). P-values are indicated as calculated by one-way ANOVA with Dunnett’s test of multiple comparisons (A-E). Source data

Extended Data Fig. 9 BC1618 increases Ampkα protein levels in mice after dietary restriction.

a, b, C57BL/6 mice were intraperitoneally (i.p.) administered with vehicle or BC1618 (20 mg/kg) on day 1 and simultaneously chow was removed for overnight starvation. After a 17 h fast, mice were given vehicle or BC1618 i.p. 30 min later, mice were euthanized, PBS was perfused through heart to remove blood. Heart, liver, and skeletal muscle was collected and fresh frozen for tissue homogenization and immunoblotting. The pAmpkα and Ampkα protein levels were quantitated and plotted in b. Densitometry data were corrected to tubulin and normalized to vehicle. Data represented mean +/- SEM (n=4-5 independent mice per group). P-values are indicated as calculated by two-tailed unpaired t-test (B). Source data

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Liu, Y., Jurczak, M.J., Lear, T.B. et al. A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance. Nat Chem Biol 17, 298–306 (2021). https://doi.org/10.1038/s41589-020-00723-0

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