Discovery of spirocyclic-diamine inhibitors of mammalian acetyl CoA-carboxylase.

A novel series of spirocyclic-diamine based, isoform non-selective inhibitors of acetyl-CoA carboxylase (ACC) is described. These spirodiamine derivatives were discovered by design of a library to mimic the structural rigidity and hydrogen-bonding pattern observed in the co-crystal structure of spirochromanone inhibitor I. The lead compound 3.5.1 inhibited de novo lipogenesis in rat hepatocytes, with an IC50 of 0.30 µM.

Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors.

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.

Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.

Pharmacologic inhibition of site 1 protease activity inhibits sterol regulatory element-binding protein processing and reduces lipogenic enzyme gene expression and lipid synthesis in cultured cells and experimental animals.

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 microM PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC(50) of 0.5 microM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

Aminopyrrolidineamide inhibitors of site-1 protease.

The discovery and efficacy of a series of potent aminopyrrolidineamide-based inhibitors of sterol regulatory element binding protein site-1 protease is described.