Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 2: Improvement of in vitro activity

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IkappaB kinase beta (IKK-beta) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring resulted in a marked increase in both kinase enzyme and cellular potencies, and provided potent IKK-beta inhibitors with IC(50) values of below 100 nM.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Polysaccharide Produced by the Genus Pullularia: I. Production of Polysaccharide by Growing Cells

A dextranlike polysaccharide was found to be produced on substrates of sucrose, maltose, glucose, and fructose by growing cells of various strains of the genus Pullularia. The polysaccharide, obtainable in amounts as large as 2 to 3 g per 100 ml of culture medium using various carbohydrates as the carbon source, was soluble in cold water but not in 50% alcohol. The polysaccharide obtained had a α_d = +197.5° (c = 0.2 in water), and its molecular weight, determined by the light-scattering method, was found to be approximately 250,000.     

Diversity in the Metabolism of Organic Acids by Pediococcus halophilus

β-N-Acetyl-d-glucosaminidase (EC 3.2.1.30) was purified from the alimentary canal of the silkworm, Bombyx mori, by ammonium sulfate fractionation and chromatography with hydroxylapatite, DEAE Bio-Gel A, chromatofocusing, and Sephacryl S-200. The purified enzyme was a single band on disc-PAGE. The molecular weight was 119,000 by gel filtration and 125,000 by SDS-PAGE. The enzyme was separated into two peptides whose apparent molecular weights were 67,500 and 57,500 by SDS-PAGE. The pi was 4.86 by chromatofocusing. The optimum pH was 5.5 to 6.0 and the optimum temperature, 45°C, using pNp-β-GlcNAc as the substrate. The enzyme was stable from pH 5.5 to 8.5 and below 30°C. It was strongly inhibited by HgCl₂. Small N-acetylchitooligomers were as good substrates as pNp-β-GlcNAc, and the enzyme cleaved colloidal chitin to GlcNAc, even though the relative velocity was slow. Smaller N-acetylchitooligomers were preferred substrates with Km 0.787 to 0.056mm, A:cat 1013 to 52sec^–1, and kcat/Km 1690 to 754 mm^–1 sec^–1. The enzyme precipitated in as band with moulting fluid chitobiase antiserum, but not with haemolymph exo-β-N-acetylglucosaminidase antiserum. The results suggest that this enzyme is an exo-type enzyme which is involved in the degradation of chitin to GlcNAc.     

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.