RESUMO
A set of focused analogues have been generated around a lead indirect adenosine monophosphate-activated kinase (AMPK) activator to improve the rat clearance of the molecule. Analogues were focused on inhibiting amide hydrolysis by the strategic placement of substituents that increased the steric environment about the secondary amide bond between 4-aminopiperidine and pyridine-5-carboxylic acid. It was found that placing substituents at position 3 of the piperidine ring and position 4 of the pyridine could all improve clearance without significantly impacting on-target potency. Notably, trans-3-fluoropiperidine 32 reduced rat clearance from above liver blood flow to 19 mL/min/kg and improved the hERG profile by attenuating the basicity of the piperidine moiety. Oral dosing of 32 activated AMPK in mouse liver and after 2 weeks of dosing improved glucose handling in a db/db mouse model of Type II diabetes as well as lowering fasted glucose and insulin levels.
Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Ratos , Animais , Proteínas Quinases Ativadas por AMP , Diamida , Glucose , Piridinas/farmacologia , Piperidinas , AmidasRESUMO
Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocôndrias Hepáticas/metabolismo , Células Musculares/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Camundongos , Mitocôndrias Hepáticas/patologia , Células Musculares/patologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
A novel small-molecule inhibitor, referred to here as R706, was discovered in a high-throughput screen of chemical libraries against Huh-7-derived replicon cells carrying autonomously replicating subgenomic RNA of hepatitis C virus (HCV). R706 was highly potent in blocking HCV RNA replication as measured by real-time reverse transcription-PCR and Western blotting of R706-treated replicon cells. Structure-activity iterations of the R706 series yielded a lead compound, R803, that was more potent and highly specific for HCV replication, with no significant inhibitory activity against a panel of HCV-related positive-stranded RNA viruses. Furthermore, HCV genotype 1 replicons displayed markedly higher sensitivity to R803 treatment than a genotype 2a-derived replicon. In addition, R803 was tested by a panel of biochemical and cell-based assays for on-target and off-target activities, and the data suggested that the compound had a therapeutic window close to 100-fold, while its exact mechanism of action remained elusive. We found that R803 was more effective than alpha interferon (IFN-alpha) at blocking HCV RNA replication in the replicon model. In combination studies, R803 showed a weak synergistic effect with IFN-alpha/ribavirin but only additive effects with a protease inhibitor and an allosteric inhibitor of RNA-dependent RNA polymerase (20). We conclude that R803 and related heterocyclic compounds constitute a new class of HCV-specific inhibitors that could potentially be developed as a treatment for HCV infection.