Metabolism and excretion of zafirlukast in dogs, rats, and mice.

The in vivo metabolism and excretion of zafirlukast [Accolate; 4, 5-cyclopentoxycarbonylamino-3-[(2-methoxy-4,2- methylphenylsulfonylaminocarbonyl)phenylmethyl]-1-methylindole], a selective peptide leukotriene receptor agonist, were investigated in mice, rats, and dogs. Leukotrienes are a class of compounds that have been identified as being responsible for the contraction of human airway and lung vascular smooth muscle. A chemical agent that is effective in blocking the induced constricting actions of leukotrienes could be used to treat inflammatory processes in the pulmonary system. Zafirlukast has been shown to be clinically efficacious and has been approved for the treatment of asthma in humans. To determine the metabolic fate of zafirlukast, the radiolabeled compound was administered orally to mice, rats, and dogs and iv to rats and dogs. Plasma, urine, and feces samples were collected, assayed for radioactivity, and profiled for metabolites. Nearly all of the [14C]zafirlukast-derived radioactivity was excreted in the feces of the test species, indicating biliary clearance as the major route of elimination from the systemic circulation. The primary routes of metabolism in all species studied involved hydrolysis of the amide linkage at the 5-aminoindole position and hydroxylation at one or more sites. Additional metabolites were formed by N-acetylation (not in dogs), demethylation of the indole nitrogen, and N-desmethylation. Accolate is a registered trademark, property of Zeneca Ltd.

Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor.

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.

The influence of amino acid sequence on the fibrillogenicity and amyloidogenicity of the carboxy-terminus of beta-amyloid precursor protein.

C-APP, a synthetic peptide corresponding to the C-terminal 20 amino acids of beta-amyloid precursor protein, forms amyloid fibrils in vitro. We investigated the effect of altering the C-APP sequence or deleting part of it on its ability to form amyloid fibrils. Substituting any single amino acid in the C-APP sequence with alanine did not prevent the formation of CAPP-like fibrils. Peptides with single or multiple substitutions that included T11, F14, F15, or Q19 showed reduced fibril-forming capacity while those with K1 and/or K13 replaced with alanine or glutamic acid showed enhanced capacity. When P10 or F14 was replaced with alanine, the fibrils were less congophilic than C-APP fibrils. All of the truncated peptides that were able to form fibrils contained at least 9 amino acids from the N-terminus of C-APP or amino acids 7-20 from the C-terminus. However, several peptides that met these criteria, but started at Q3 or contained only 2-4 amino acids C-terminal to P-10, failed to form many or typical fibrils. Peptides that contained the C-APP sequence plus 5-20 adjacent amino acids from the beta-amyloid precursor protein formed fibrils less readily than C-APP and most of the fibrils were not congophilic. The exception was CAPP-30, which formed moderate amounts of congophilic fibrils resembling C-APP fibrils morphologically. Therefore, proteolysis which releases C-APP from these peptides (except CAPP-30) would be predicted to enhance their amyloidogenicity. These results suggest that several features of C-APP peptide may be important in fibril formation. One of these features is the length of the peptide, with lengths of about 10, 20, or 30 amino acids, favoring fibril formation.

Isolation and pharmacological characterization of omega-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses.

omega-Grammotoxin SIA, a peptidergic blocker of voltage-sensitive calcium channel (VSCC) responses, was purified from Grammostola spatulata (tarantula spider) venom by reverse phase high performance liquid chromatography. Protease-sensitive biological activity was monitored by determining the inhibition of K(+)-stimulated influx of 45Ca2+ into rat brain synaptosomes. Electrospray mass spectrometry indicated an average molecular mass of 4109.2 Da for the native peptide. Chemical reduction of omega-grammotoxin SIA indicated the presence of three disulfide bridges. Primary sequence data confirmed the existence of six cysteine residues and 36 residues in total, with an average theoretical molecular mass of 4109.7 Da for the amidated carboxyl-terminal species. The biological profile of omega-grammotoxin SIA indicated virtually complete blockade of presynaptic vertebrate N-type as well as P-type VSCC responses. Specifically, omega-grammotoxin SIA caused a concentration-dependent and virtually complete inhibition of K(+)-evoked influx of 45Ca2+ into either rat or chick brain synaptosomes. Similar inhibition profiles were generated for the inhibition of release of either D-[3H]aspartate or [3H]norepinephrine from rat hippocampal or [3H]norepinephrine from chick cortical brain slice preparations evoked by K+ depolarization. As reported earlier, omega-grammotoxin SIA did not inhibit 125I-omega-conotoxin GVIA, [3H]PN 200-110, or [3H]desmethoxyverapamil binding to neuronal membrane fragments. To our knowledge, omega-grammotoxin SIA is the first ligand identified to block putative N-channel function without displacement of 125I-omega-conotoxin GVIA. omega-Grammotoxin SIA thus represents a novel vertebrate VSCC antagonist that inhibits neuronal N- and P-type VSCC responses.

Effects of site-specific acetylation on omega-conotoxin GVIA binding and function.

Chemical modification of omega-conotoxin GVIA (omega-CgTXGVIA) was performed using nonsaturating concentrations of acetic anhydride to generate seven distinct derivatives. Following separation of these peptides using reverse-phase HPLC (RP-HPLC), their individual molecular weights were determined using fast bombardment mass spectrometry (FAB-MS). Three peptides contained a single acetylated amino group, three possessed two acetylated amino groups, and the last contained three acetylations. For each peptide, the specific site of acetylation was confirmed using a scheme of tryptic digestion, under nonreducing conditions, followed by RP-HPLC and FAB-MS. Biological profiles for each peptide were obtained by analyzing their capacity to displace native 125I-omega-CgTx GVIA binding to rat hippocampal membranes and to block K(+)-stimulated 45Ca2+ influx into chick brain synaptosomes. The data indicate that successive additions of acetyl moieties to omega-CgTx GVIA lead to a loss of both binding affinity and Ca2+ influx inhibitory potency. Within the monoacetylated series, acetylation of the amino terminal of Cys-1, as compared to the epsilon-amino group of either Lys-2 or Lys-24, leads to the greatest shift in potency. In summary, these results indicate that basic (i.e., primary amino) groups, which are brought into close proximity as a result of disulfide bridging, are important in the functional blockade of neuronal Ca2+ channels by omega-CgTx GVIA.

Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and effect on microtubule assembly.

Tau protein is an integral component of paired helical filaments, a pathological feature of Alzheimer's disease. tau extracted from these filaments displays decreased electrophoretic mobility due to aberrant phosphorylation. Here we show that recombinant human tau can be phosphorylated by cAMP-dependent protein kinase resulting in decreased electrophoretic mobility. Phosphorylation of tau by cAMP-dependent protein kinase caused a 92% decrease in the maximum rate of tau-induced microtubule assembly. The sites of phosphorylation were identified by digesting phosphorylated tau with proteases, separating the peptides by reversed-phase HPLC, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase N-terminal sequencing. Five phosphorylation sites were identified, two of which were located within microtubule binding domains. One site was previously shown to be the sole phosphorylation site for CaM kinase II; phosphorylation at this site by CaM kinase II was sufficient to cause decreased electrophoretic mobility (Steiner, B., Mandelkow, E. M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Soling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) EMBO J. 9, 3539-3544). Thus two different second messenger-dependent protein kinases can phosphorylate tau at the same site and induce a shift in tau mobility like that seen in Alzheimer's disease.