Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.

Cytotoxic amide alkaloids from Piper boehmeriaefolium.

Eight new amide alkaloids (1-8) and 19 known ones were isolated from the whole plant of Piper boehmeriaefolium. Their structures were determined through spectroscopic data analyses. Cytotoxic activity of these amides against human cervical carcinoma HeLa cells was evaluated, and 1-[(9E)-10-(3,4-methylenedioxyphenyl)-9-decenoyl]pyrrolidine (9) exhibited significant inhibitory activity with an IC(50) value of 2.7 µg/mL.

Novel small-molecule AMP-activated protein kinase allosteric activator with beneficial effects in db/db mice.

AMP-activated protein kinase (AMPK) is an energy sensor of metabolism that is an attractive therapeutic target for type 2 diabetes mellitus and metabolic syndrome. Using a homogeneous scintillation proximity assay (SPA), we identified a new small-molecule AMPK activator, ZLN024, which allosterically stimulated active AMPK heterotrimers and the inactive α1 subunit truncations α1 (1-394) and α1 (1-335) but not α1 (1-312). AMPK activation by ZLN024 requires the pre-phosphorylation of Thr-172 by at least one upstream kinase and protects AMPK Thr-172 against dephosphorylation by PP2Cα. ZLN024 activated AMPK in L6 myotubes and stimulated glucose uptake and fatty acid oxidation without increasing the ADP/ATP ratio. ZLN024 also activated AMPK in primary hepatocytes, decreased fatty acid synthesis and glucose output. Treatment of db/db mice with 15 mg/kg/day ZLN024 improved glucose tolerance; liver tissue weight, triacylglycerol and the total cholesterol content were decreased. The hepatic transcriptional level of G6Pase, FAS and mtGPAT were reduced. The transcription of genes involved in fatty acid oxidation and the mitochondrial biogenesis of muscle tissue were elevated. The ACC phosphorylation was increased in muscle and liver. This study provides a novel allosteric AMPK activator for functional study in vitro and in vivo and demonstrates that AMPK allosteric activators could be a promising therapeutic approach for type 2 diabetes mellitus and metabolic syndrome.

AMPK activators as novel therapeutics for type 2 diabetes.

AMPK is a potential target of metabolic diseases including obesity and type 2 diabetes. The activation of AMPK can lead to an increase of glucose uptake into muscle, decreased gluconeogenesis in liver, increased fatty acid oxidation in muscle and liver, decreased fatty acid synthesis in liver and adipose tissue, and increase mitochondrial biogenesis. Until now, many efforts from industrial and academia have been focused on searching novel agents that activate AMPK directly or indirectly. This review will discuss recent advances in the search for novel therapeutic agents that mediate their activity via AMPK activation.

High-throughput assay for modulators of mitochondrial membrane potential identifies a novel compound with beneficial effects on db/db mice.

OBJECTIVE: Recently, several drugs have been shown to exert beneficial effects for metabolic syndrome through mild regulation of mitochondrial function. Hence, we explored a strategy of targeting mitochondrial function to improve glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: Mitochondrial membrane potential (Deltapsim) is a marker of mitochondrial function; therefore, we set up a high-throughput screening assay of Deltapsim in L6 myotubes. The effects of a selected lead compound were investigated in vitro and in vivo in relation to metabolic syndrome. RESULTS: A novel small-molecule compound, C1, was identified through this high-throughput screening. C1 depolarized Deltapsim in L6 myotubes without cytotoxicity and led to increased cellular AMP-to-ATP ratio, activation of AMP-activated protein kinase (AMPK), and enhanced glucose uptake. It also stimulated the AMPK pathway in HepG2 cells, leading to decreased lipid content. Intriguingly, C1 inhibited respiration in L6 myotubes but did not affect respiration in isolated muscle mitochondria, suggesting that it may depolarize Deltapsim indirectly by affecting the supply of electron donors. Acute administration of C1 in C57BL/6J mice markedly increased fat oxidation and the phosphorylation of AMPK and acetyl-CoA carboxylase in the liver. In diabetic db/db mice, chronic administration of C1 significantly reduced hyperglycemia, plasma fatty acids, glucose intolerance, and the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver. CONCLUSIONS: Our results demonstrate a novel small molecule that mildly depolarizes Deltapsim and is able to improve glucose and lipid metabolism to exert beneficial effects for metabolic syndrome. These findings suggest that compounds regulating mitochondrial function may have therapeutic potential for type 2 diabetes.

Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells.

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.

Isoquinoline-1,3,4-trione derivatives inactivate caspase-3 by generation of reactive oxygen species.

Caspase-3 is an attractive therapeutic target for treatment of diseases involving disregulated apoptosis. We report here the mechanism of caspase-3 inactivation by isoquinoline-1,3,4-trione derivatives. Kinetic analysis indicates the compounds can irreversibly inactivate caspase-3 in a 1,4-dithiothreitol (DTT)- and oxygen-dependent manner, implying that a redox cycle might take place in the inactivation process. Reactive oxygen species detection experiments using a chemical indicator, together with electron spin resonance measurement, suggest that ROS can be generated by reaction of isoquinoline-1,3,4-trione derivatives with DTT. Oxygen-free radical scavenger catalase and superoxide dismutase eliciting the inactivation of caspase-3 by the inhibitors confirm that ROS mediates the inactivation process. Crystal structures of caspase-3 in complexes with isoquinoline-1,3,4-trione derivatives show that the catalytic cysteine is oxidized to sulfonic acid (-SO(3)H) and isoquinoline-1,3,4-trione derivatives are bound at the dimer interface of caspase-3. Further mutagenesis study shows that the binding of the inhibitors with caspase-3 appears to be nonspecific. Isoquinoline-1,3,4-trione derivative-catalyzed caspase-3 inactivation could also be observed when DTT is substituted with dihydrolipoic acid, which exists widely in cells and might play an important role in the in vivo inactivation process in which the inhibitors inactivate caspase-3 in cells and then prevent the cells from apoptosis. These results provide valuable information for further development of small molecular inhibitors against caspase-3 or other oxidation-sensitive proteins.

Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits.

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.

Construction of a small peptide library related to inhibitor OM99-2 and its structure-activity relationship to beta-secretase.

AIM: To develop probes for detecting the binding specificity between beta-secretase and substrate, and provide reliable biological activity data for further researching encircling substrate-based inhibitors. METHODS: To prepare the inhibitors, the hydroxyethylene (HE) segment including P1 and P1'was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human beta-secretase ectodomain (amino acid residues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this beta-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and molecular mechanics-generalized-born surface area (MM-GBSA) analysis, were performed on the complex of beta-secretase and OM99-2 to obtain the geometrical and energetical information. RESULTS: We designed and constructed a positional scanning combinatorial library including 16 compounds; all members of the library were synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P4-P1 and P1' -P4'positions led to the discoveries of P and P'sides binding specificity and potent inhibitors 14, 18, and 22. The binding free energy on the whole system and every residue were compared to the biological assay result. CONCLUSION: The removal of P4' yielded inhibitor 22 (A3 *B2) with high potency; further truncation of P3'gave inhibitor 18 (A3 *B1) with equal activity, implying that the right side of the inhibitors play a less important role and could be easily simplified, while change on the P side may cause substantial results.