Adenosine monophosphate-activated protein kinase as a potential target for synthetic chalco-naringenin analogs and putative therapeutic applications.

Abstract

Introduction: Diabetes mellitus is a multifactorial chronic metabolic disorder that is characterized by defects in endogenous insulin secretion or action, or both, resulting in chronic hyperglycemia, a clinical hallmark of diabetes. Metformin is currently the first-line drug of choice for the treatment of type 2 diabetes (T2D), being prescribed to at least 120 million people worldwide. It exerts its antidiabetic effects by reducing hepatic glucose production and increased peripheral glucose utilization through activation of AMP-activated protein kinase (AMPK). However, despite significant gains with metformin as a monotherapy in T2D, some patients experience gastrointestinal disturbances and lactic acidosis although the latter is very rare. Moreover, some patients still fail to achieve optimum glycemic control when treated on metformin only. Naringenin, a flavonoid exerts its antidiabetic effects by inhibition of gluconeogenesis through upregulation of AMPK, hence metformin-like effects. Because of these similar pharmacological effects between naringenin and metformin, our laboratory synthesized analogs of chalco-naringenin compounds which could be more permeable to the plasma membrane and hence putatively increased pharmacological effects. Aims: To identify AMPK as a potential target of synthetic chalco-naringenin analogs and putative therapeutic applications. Methods: A novel series of 4-[(cyclopropylcarbonyl)amino] chalco-naringenin analogs, compound 5a to 5k, were synthesized and characterized by IR, 1H-NMR and 13C-NMR. In silico screening of the compounds was conducted to evaluate potential antidiabetic activity of the novel chalco-naringenin series. Compounds 2-chlorophenyl (5b) and 5k (2,3-dimethoxyphenyl) had highest binding affinity to AMPK hence were chosen for a study. C2C12 and Chang cells were cultured in dulbecco's modified eagle medium (DMEM) and eagle's minimum essential medium (EMEM) media, respectively, allowed to grow to 80% confluence, and then exposed to different concentrations. MTT assay was used to determine cell viability and chalcones were subjected for 12, 24 and 48 hours at concentrations (10-750 μM). Cells were exposed to metformin (2-10 mM), naringenin (50-500 μM) and chalcones (10-500 μM) for 48 hours and further subjected to phospho-AMPKα (Thr172) sandwich ELISA Kit to determine phosphorylation of AMPK. To measure the amount of glycogen in cells after exposure to metformin, naringenin and chalcones for 48 hours, cells were harvested (1x106 per mL) and the glycogen assay performed according to Seifter et al. (1950). Results: A chalcone series of eleven compounds were successfully synthesized using the Claisen-Schmidt reaction. The absorbance values and peaks observed on the IR spectra confirmed the different functional groups observed on the compounds. The synthesized compounds were also characterized through 13C-NMR and 1H-NMR spectroscopy. The 13C-NMR spectras indicated the presence of the CH2 group of the cyclopropylcarbonyl amide and there were certain distinct peaks on the spectras that identify carbon atoms found on the compounds. In 1H-NMR, the chemical shift for all the CH2 groups on the cycloalkane resonated around δ 1,16 – 0,80 ppm as multiplets, while the other CH multiplet resonated around δ 1,62 – 1,58 ppm. Docking scores of the chalco-naringenin series suggested a good binding affinity of these compounds to AMPK, with compound 5b showing the highest binding affinity to AMPK. Cell viability as determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays were found to be dose-dependent for all compounds and compound 5k exerted reduced cell viability as compared to 5b. Furthermore, compound 5b presented with higher IC50 values compared to 5k. The effects of chalcones on AMPK phosphorylation were potentiated by co-treatment with metformin or naringenin. Metformin, naringenin and chalcone 5b significantly reduced synthesis of glycogen as compared to control (p < 0.05). Conclusions: Chalco-naringenin analogs showed potential in expression of AMPK through computational chemistry, however in the in vitro model the effects of chalcones on AMPK were potentiated by metformin and naringenin. The chalcones could further be explored for their potential on AMPK activity on primary hepatocytes and/ or in vivo studies.