Methods Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes.Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion (18). For the AMPK assay, cells were seeded in six-well plates at 1.5 × 10 6 cells/well in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 µg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide ribo-

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin's antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation-deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

Finasteride is employed in treatment of benign prostatic hyperplasia in man, where its target enzyme is steroid 5α-reductase. It is a novel, potent mechanism-based inhibitor of the human prostate (type 2) isozyme. Although it is accepted as an alternate substrate and is ultimately reduced to dihydrofinasteride, this proceeds through an enzyme-bound NADP−dihydrofinasteride adduct. Finasteride is processed with a second-order rate constant, k i/K i = 1 × 106 M-1 s-1, that approaches k cat/K m for reduction of testosterone, 3 × 106 M-1 s-1, and essentially every catalytic event is lethal (partition ratio ≤ 1.07). The membrane-bound enzyme−inhibitor complex formed from [3H]finasteride appears to release [3H]dihydrofinasteride with a half-life of 1 month at 37 °C (k = (2.57 ± 0.03) × 10-7 s-1), as identified by mass spectroscopy. The intermediate NADP−dihydrofinasteride adduct can be recovered intact by denaturation of the enzyme−inhibitor complex and has been purified. Free in solution, it likewise decomposes to dihydrofinasteride (half-life = 11 days). An extremely potent bisubstrate analog inhibitor, this NADP−dihydrofinasteride adduct binds to the free enzyme with a second-order rate constant equal to k cat/K m for turnover of testosterone and has a dissociation constant K i ≤ 1 × 10-13 M. Finasteride is also a mechanism-based inhibitor of the human skin (type 1) isozyme, but it is processed with a much smaller second-order rate constant, k i/K i = 3 × 103 M-1 s-1, which attenuates its activity against this isozyme in vivo. The mechanism explains the exceptional potency and specificity of finasteride in treatment of benign prostatic hyperplasia, and the concept may have application to other pyridine nucleotide-linked enzymes.