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.

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).

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).

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).

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).

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).

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).

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

(A) THP1 cells stably expressing an NF-κB inducible luciferase (secretary) 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 × 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).

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).

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).

Fig. 1.. pAmpkα undergoes SCF^Fbxo48 mediated ubiquitin proteasomal degradation.

(A) BEAS-2B cells were pretreated with glucose free DMEM 2% media, including DMSO alone (control, Ctrl), with Carfilzomib ([CFZ], 1 μM) or leupeptin (20 μM) for 30 min, then CHX (40 μg/ml) was added for additional 30 min, cells were collected for immunoblot analysis (Results are representative of three independent experiments). (B) BEAS-2B cells were pretreated in glucose free media with or without MG132 (20 μM) for 1 h. Whole cell extracts were subjected to TUBE agarose beads pull down (PD) and immunoblotting. Cells in glucose-rich full media served as a negative control. Arrows indicate polyubiquitylated products. Ub: ubiquitin (Results are representative of three independent experiments). (C) BEAS-2B cells were cultured in glucose free DMEM 2% media at indicated times. Whole cell lysates were subjected to immunoblotting (Results are representative of three independent experiments). (D) BEAS-2B cells were nucleofected with increasing amounts of V5 tagged Fbxo48 plasmids. After 60 h incubation, cells were then exposed to 2% DMEM media without glucose before immunoblotting (Results are representative of three independent experiments). (E) BEAS-2B cells were nucleofected with empty vector or Fbxo48-V5 plasmid. 48 h later, cell lysates were subjected to immunoprecipitation (IP) using V5 antibodies and IP output was assayed with immunoblotting (Results are representative of two independent experiments). (F) Biotin labeled Ampkα peptides were first bound to streptavidin beads. Ampk beads were then incubated with purified recombinant Fbxo48 protein for 2 h. Beads were then washed, and protein was eluded before immunoblotting (Results are representative of two independent experiments). (G) BEAS-2B cells were nucleofected with 2 μg of either empty vector or Fbxo48-V5 plasmid. 48 h later, cells were exposed to glucose free 2% DMEM media for 1 h, and then CHX (40 μg/ml) was added at indicated time points. Cells were collected for immunoblotting (Results are representative of two independent experiments). (H) BEAS-2B cells were nucleofected with control or Fbxo48 siRNA. After 72 h incubation, cells were treated with CHX and assayed for immunoblot (Results are representative of two independent experiments). (I) BEAS-2B cells were nucleofected with 50 pg control or Fbxo48 siRNA. After 72 h incubation, cells were treated in glucose free media for a time course before assayed for immunoblotting (Results are representative of three independent experiments).

Fig. 2.. Small molecule Fbxo48 inhibitors increase pAmpkα.

(A) BEAS-2B cells were incubated in glucose free 2% DMEM media for 16 h containing AICAR at indicated concentrations, with or without 4 μM BC1583. Non-treated cells cultured in glucose containing full media served as a negative control. Cell lysates were subjected to immunoblotting (Results are representative of three independent experiments). (B) BEAS-2B cells were cultured in glucose free 2% DMEM media overnight with various concentrations of BC1583 before immunoblotting (Results are representative of four independent experiments). (C) The hit compound BC1583 and its analog BC1618. Red circles illustrate the essential signatures of the molecule. (D) BEAS-2B cells were pretreated with BC1618 (10 μM) for 2 h in full media. Media were then switched to glucose free 2% DMEM media for additional 1 h incubation. CHX (40 μg/ml) was then added at indicated time points before immunoblotting (Results are representative of three independent experiments). (E) BEAS-2B cells were incubated in glucose free 2% DMEM media containing either BC1618 or metformin at indicated concentrations for 16 h. Cell were collected for immunoblotting (Results are representative of five independent experiments).

Fig. 3.. Fbxo48 inhibitor interrupts Fbxo48/pAmpkα interaction.

(A) 10 μg phospho-Ampkα peptide (Biotin-DGEFLRT(p)SCGSPN) was used as bait for recombinant Fbxo48 capture and PD with streptavidin in the presence of BC1618 titration (Results are representative of three independent experiments). (B) 1 μg recombinant Fbxo48 (HIS-tagged) protein was used as bait for recombinant pAmpkα capture and PD with HIS beads in the presence of BC1618 titration (Results are representative of three independent experiments). (C) Cell based thermal shift assay (CETSA) of Fbxo48-V5 and Fbxo30-V5 proteins in HEK293 cells treated with BC1618 (3μM) or DMSO (Results are representative of three independent experiments). The graphs below show differential ability of BC1618 to raising the thermal stability curve of Fbxo48 unlike control Fbxo30. (D) BEAS-2B cells were nucleofected with 50 pg control siRNA or Fbxo48 siRNA. After 72 h incubation, cells were exposed to BC1618 in a concentration dependent manner in glucose free 2% DMEM media for 6 h before immunoblotting (Results are representative of three independent experiments).

Fig. 4.. BC1618 facilitates mitochondria fission and autophagy.

(A) BEAS-2B cells were exposed to either DMSO (vehicle, Veh) or BC1618 (10 μM) in 2% DMEM media with or without glucose for 5 h. Cells were then stained with 100 nM Mitotracker Green FM for 25 min before confocal microscopy. Scale bar=10 μm (Results are representative of two independent experiments). (B) BEAS-2B cells were exposed to BC1618 at indicated concentrations in glucose free 2% DMEM media for 4 h before immunoblotting. Cells in high glucose media served as controls (Results are representative of three independent experiments). (C) BEAS-2B cells were incubated in glucose free 2% DMEM media containing BC1618 at indicated concentrations for 6 h before immunoblotting. Cells in high glucose media served as controls (Results are representative of three independent experiments). (D) BEAS-2B cells were exposed to BC1618 in glucose free 2% DMEM media with or without Bafilomycin A1 (Baf, 50 nM) for 4 h before immunoblotting. Non-treated cells in high glucose media represented the control group (Results are representative of two independent experiments). (E) HEK293A cells stably expressing GFP-LC3 were incubated in 2% high glucose DMEM media containing Baf (2.5 nM) with or without BC1618 for 24 h. Cells were fixed in 4% paraformaldehyde/PBS for 15 min before DAPI staining. Cells were then subjected to confocal microscopic analysis (Results are representative of three independent experiments). Scale bar = 10 μm. (F) Puncta from 50 randomly picked cells of each group from (E) were counted and plotted. Box plot quantification represent median and interquartile range with P-value range indicated by one-way ANOVA with Tukey’s test of multiple comparisons (F).

Fig. 5.. BC1618 improves hepatic insulin sensitivity in diet-induced obese mice.

(A) Plasma glucose levels (upper panel) and the glucose infusion rate (GIR; lower panel) required to maintain euglycemia during a hyperinsulinemic infusion. Data are mean ± s.e.m. for n=6 per group; 80 min, p=0.0390; 90 min, p=0.0344; 100min, p=0.0384; 110min, p=0.0398; 120min, p=0.0398, as determined by two-way ANOVA with Sidak’s test of multiple comparisons. (B) GIR during steady-state or the last 40 min of the 120 min study, data are mean ± s.e.m. for n=6 per group and were compared by unpaired, two-tailed Student’s t-test.(C) Rates of whole-body insulin-stimulated glucose uptake during steady-state, data are mean ± s.e.m. for n=6 per group and were compared by unpaired, two-tailed Student’s t-test.(D) Rates of basal and insulin-stimulated endogenous or hepatic glucose output (EGP) during the study, data are mean ± s.e.m. for n=6 per group and were compared by unpaired, two-tailed Student’s t-test. (E) Basal and clamped plasma insulin levels, data are mean ± s.e.m. for n=6 per group and were compared by unpaired, two-tailed Student’s t-test.