Development of novel adenosine monophosphate-activated protein kinase activators

Abstract

In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)gamma-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homologue of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively) and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoro-methyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event.

Figure 1

(A) Schematic representation of the two-tiered screening of the benzylidene-thiazolidinedione-based focused compound library to identify lead AMPK activators. (B) General synthetic procedure for Series A – C compounds. Reaction conditions: Series A: a, K₂CO₃/R1-Br; b, LAH, THF; c, (CF₃SO₂)₂O, pyridine, CH₂Cl₂; d, K₂CO₃, DMF; e, AcOH, piperidine, ethanol/reflux. Series B: a, AcOH, piperidine, ethanol/reflux; b, K₂CO₃, DMF. Series C: a, K₂CO₃, DMF; b, pyridine, CH₂Cl₂; c, LAH, dry THF, 0°C; d, MnO₂, CH₂Cl₂, reflux; d, piperidine, EtOH, reflux; e, AcOH, piperidine, ethanol/reflux.

Figure 2

Chemical structures of compounds 1 – 60 in the thiazolidinedione-based focused compound library.

Figure 3

(A) Schematic representation of the role of AMPK as a negative regulator of mTOR-and IL-6/IL-6 receptor-mediated signaling pathways. (B) Western blot analysis of the effects of ciglitazone and 62, each at 10 µM, on the phosphorylation of AMPK, p70S6K, and Stat3 in LPS-treated THP-1 cells relative to that on LPS-treated and untreated (Ctr) THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. (C) Left panel, ELISA analysis of the inhibitory effects of ciglitazone (CG) and 62 at the indicated concentrations on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). Right panel, the corresponding effects on the viability of THP-1 cells by MTT assays (N = 6).

Figure 4

(A) Western blot analysis of the effects of compounds 1 – 60 vis-à-vis ciglitazone (CG), each at 10 µM, on the phosphorylation of AMPK, p70S6K, and Stat3 in LPS-treated THP-1 cells relative to that in LPS only-treated (L) and untreated (Ctr) THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. (B) Upper panel, ELISA analysis of the inhibitory effects of compounds 1 – 60 vis-à-vis ciglitazone (CG), each at 10 µM, on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). Lower panel, the corresponding effects on the viability of THP-1 cells by MTT assays (N = 6).

Figure 5

(A) Effects of compounds 8, 12, 31, 44, 49, 53 and 54 vis-à-vis ciglitazone (CG), each at 10 µM, on PPARγ activation in differentiated THP-1 cells. THP-1 cells were transiently transfected with the PPRE-x3-TK-Luc reporter vector and then exposed to individual agents or DMSO vehicle in 10% FBS-supplemented RPMI 1640 medium for 48 h. Analysis of luciferase activity was carried out as described in the Experimental Section. Columns, mean; bars, SD (N = 6). (B) Upper panel, ELISA analysis of the inhibitory effects of compounds 8, 12, 21, 31, 44, 49, 53 and 54 vis-à-vis ciglitazone (CG), each at 1 µM, on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). Lower panel, the corresponding effects on the viability of THP-1 cells by MTT assays (N = 6).

Figure 6

(A) ELISA analysis of the dose-dependent effect of compound 53 on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). (B) RT-PCR analysis of the dose-dependent suppressive effect of compound 53 on the mRNA levels of IL-6 in LPS-treated THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). (C) Western blot analysis of the dose-dependent effect of compound 53 relative to 0.5 mM AICAR on the phosphorylation levels of AMPK and p70S6K in LPS-treated THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment.

Figure 7

(A) Western blot analysis of the expression levels of AMPK in THP-1 macrophages transiently transfected with the dominant negative (DN)-AMPK plasmid or pCMV empty vector. (B) Protective effect of ectopic expression of DN-AMPK on LPS-stimulated IL-6 production in THP-1 macrophages with or without co-treatment with 10 µM compound 53. Columns, mean; bars, SD (N = 3). (C) Western blot analysis of the dose- and time-dependent effects of compound 53 on the phosphorylation levels of AMPK and p70S6K in C-26 adenocarcinoma cells in 10% FBS-containing medium. (D) The surface electrostatic potentials and structures of compound 53 versus compounds 63 and 64.