ABSTRACT
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.
ABSTRACT
PURPOSE: Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments. METHODS: We designed and synthesized MDAP(1000), MDAP(3000), and MDAP(5000), which consist of 4 independent moieties: RI unit (111)In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates. RESULTS: MDAP(1000), MDAP(3000), and MDAP(5000) were cleaved by MMP-2 in a concentration-dependent manner. MDAP(3000) pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP(3000) exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP(3000) exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP(3000) existed in tumor xenografts in vivo. CONCLUSIONS: The results indicate the possible usefulness of our MDAP strategy for tumor imaging.
