4-Aminoquinoline melanin-concentrating hormone 1-receptor (MCH1R) antagonists.

Structure-activity relationships of a 4-aminoquinoline MCH1R antagonist lead series were explored by synthesis of analogs with modifications at the 2-, 4-, and 6-positions of the original HTS hit. Improvements to the original screening lead included lipophilic groups at the 2-position and biphenyl, cyclohexyl phenyl, and hydrocinnamyl carboxamides at the 6-position. Modifications of the 4-amino group were not well tolerated.

Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core.

We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B). High level expression in Escherichia coli has been achieved with yields of up to 20mg/L. The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand. PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM. Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B. The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674. Constructs starting at E665 and M674 were fully active and devoid of activity, respectively. A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected. This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667. Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.

The role of tryptophan 1072 in human PDE3B inhibitor binding.

The catalytic domain of recombinant human PDE3B was expressed in Escherichia coli as inclusion bodies and refolded to form active enzyme. A mutation at tryptophan 1072 in PDE3B disrupts inhibitor binding, but has minimal effect on cAMP hydrolysis. The W1072A mutation caused a 158-fold decrease in affinity for cilostamide, a 740-fold decrease for cGMP, and a 15-fold decrease in affinity for IBMX. The corresponding tyrosine mutation had a smaller effect. However, the K(m) of cAMP for the W1072A mutation was only increased by about 7-fold. The data indicate that the inhibitor binding region is not completely coincident with the substrate binding region. The homologous residue in PDE4B is located on helix 16 within 7A of the predicted bound substrate. A model of PDE3B was constructed based on the X-ray crystal structure of PDE4B.

Characterization of neuropeptide Y (NPY) Y5 receptor-mediated obesity in mice: chronic intracerebroventricular infusion of D-Trp(34)NPY.

To clarify the role of the neuropeptide Y (NPY) Y5 receptor subtype in energy homeostasis, the effect of the intracerebroventricular infusion of a selective Y5 agonist, D-Trp(34)NPY, was investigated in C57BL/6J mice. Intracerebroventricular infusion of D-Trp(34)NPY (5 and 10 microg/d) produced hyperphagia and body weight gain, accompanied by increased adipose tissue weight, hypercholesterolemia, hyperinsulinemia, and hyperleptinemia. Oral administration of a selective Y5 antagonist at a dose of 100 mg/kg twice a day completely suppressed all of these D-Trp(34)NPY-induced changes, indicating that chronic activation of the Y5 receptor produces hyperphagia and obesity. In addition, D-Trp(34)NPY still resulted in an increase in adipose tissue weight accompanied by hyperleptinemia and hypercholesterolemia, although D-Trp(34)NPY-induced food intake was restricted by pair-feeding. Under the pair-fed condition, D-Trp(34)NPY decreased hormone-sensitive lipase activity in white adipose tissue and uncoupling protein-1 mRNA expression in brown adipose tissue. These findings indicate that Y5-mediated obesity may involve metabolic changes, such as decreased lipolysis and thermogenesis, as well as hyperphagia. Therefore, the Y5 receptor can play a key role in regulating energy homeostasis.

Novel sesquiterpenoids from the fermentation of Xylaria persicaria are selective ligands for the NPY Y5 receptor.

Neuropeptide Y (NPY) is a polypeptide found in the peripheral and central nervous system and is involved in the regulation of feeding. Antagonists of NPY receptor activation could therefore have potential for development as antiobesity drugs. Fermentation of an isolate of Xylaria persicaria yielded two novel eremophilane sesquiterpenoids xylarenals A (1) and B (2). These compounds are selective for the NPY Y5 receptor but have only modest affinity. The isolation, structure elucidation, and biological activities of these compounds are described.