Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a potential therapeutic target for various medical conditions. We here identify a small-molecule compound (RX-375) that activates AMPK and inhibits fatty acid synthesis in cultured human hepatocytes. RX-375 does not bind to AMPK but interacts with prohibitins (PHB1 and PHB2), which were found to form a complex with AMPK. RX-375 induced dissociation of this complex, and PHBs knockdown resulted in AMPK activation, in the cultured cells. Administration of RX-375 to obese mice activated AMPK and ameliorated steatosis in the liver. High-throughput screening based on disruption of the AMPK-PHB interaction identified a second small-molecule compound that activates AMPK, confirming the importance of this interaction in the regulation of AMPK. Our results thus indicate that PHBs are previously unrecognized negative regulators of AMPK, and that compounds that prevent the AMPK-PHB interaction constitute a class of AMPK activator.

Quinolone derivatives containing strained spirocycle as orally active glycogen synthase kinase 3ß (GSK-3ß) inhibitors for type 2 diabetics.

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3ß inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3ß inhibitory activity in both cell-free and cell-based assays (IC(50) = 36nM, EC(50) = 3.2µM, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice.

Design, synthesis, and structure-activity relationship studies of novel 2,4,6-trisubstituted-5-pyrimidinecarboxylic acids as peroxisome proliferator-activated receptor gamma (PPARgamma) partial agonists with comparable antidiabetic efficacy to rosiglitazone.

A series of novel 2,4,6-trisubstitutedpyrimidine-5-carboxylic acid derivatives were designed and synthesized with the intent of producing a peroxisome proliferator-activated receptor gamma (PPARgamma) partial agonist for antidiabetic agents. A pharmacophore-driven approach of in-house screening identified compound 7, which led to the identification of compound 9 featuring a 2,4,6-trisubstituted pyrimidine-5-carboxylic acid core. Structure-activity relationship studies of 9 resulted in identifying 4,6-bisbenzylthio-2-methylthiopyrimidine-5-carboxylic acid (50) as the most attractive of all the screened compounds. The X-ray cocrystal structure of 50 bound on PPARgamma revealed that the key hydrogen bond interactions, which are not related to the activation function 2 (AF-2) site, are different from those of the full agonist. Compound 50 showed typical PPARgamma partial agonist properties in the PPARgamma-GAL4 functional assay and weaker differentiation of adipocytes in 3T3-L1 cells than observed with rosiglitazone. Furthermore, 50 displayed comparable antidiabetic efficacy with rosiglitazone in db/db mice, although its potency is 10-fold weaker than that of rosiglitazone.

Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.

Transcriptomic and proteomic analysis of a 14-3-3 gene-deficient yeast.

BMH1 and BMH2 encode Saccharomyces cerevisiae 14-3-3 homologues whose exact functions have remained unclear. The present work compares the transcriptomic and proteomic profiles of the wild type and a BMH1/2-deficient S. cerevisiae mutant (bmhDelta) using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis. It is reported here that, although the global patterns of gene and protein expression are very similar between the two types of yeast cells, a subset of genes and proteins (a total of 220 genes) is significantly induced or reduced in the absence of Bmh1/2p. These genes include approximately 60 elements that could be linked to the reported phenotypes of the bmhDelta mutant (e.g., accumulation of glycogen and hypersensitivity to environmental stress) and/or could be the potential downstream targets of interacting partners of Bmh1/2p such as Msn2p and Rtg3p. Importantly, >30% of the identified genes (71 genes) were found to be associated with carbon (C) and nitrogen (N) metabolism and transport, thereby suggesting that Bmh1/2p may play a major role in the regulation of C/N-responsive cellular processes. This study presents the first comprehensive overview of the genes and proteins that are affected by the depletion of Bmh1/2p and extends the scope of knowledge of the regulatory roles of Bmh1/2p in S. cerevisiae.