Aclidinium bromide is a novel potent, long-acting inhaled muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease. Aclidinium showed subnanomolar affinity for the five human muscarinic receptors (M 1 -M 5 3 H]aclidinium at the M 2 receptor was shorter than at the M 3 receptor, demonstrating kinetic selectivity for the M 3 receptor. In isolated guinea pig trachea, aclidinium showed comparable potency to ipratropium and tiotropium, faster onset of action than tiotropium, and duration of action similar to tiotropium and significantly longer than ipratropium. Nebulized aclidinium inhibited bronchoconstriction induced by acetylcholine in guinea pigs in a concentrationdependent manner with an onset of action faster than tiotropium. Duration of action of aclidinium (t 1/2 ϭ 29 h) was much longer than ipratropium (8 h) but shorter than tiotropium (64 h). In dogs, aclidinium induced a smaller and more transient increase in heart rate than tiotropium at comparable supratherapeutic doses. Therefore, under these conditions, aclidinium showed a greater therapeutic index than tiotropium (4.2 versus 1.6). These results indicate that aclidinium is a potent muscarinic antagonist with a fast onset of action, a long duration of effect, and a favorable cardiovascular safety profile.Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease characterized by chronic airflow obstruction attributed to long-term exposure to inhaled noxious gases and particles, most often related to cigarette smoking that is not fully reversible after bronchodilator therapy (www.goldcopd.org) (Rabe et al., 2007). Recent projections from the World Health Organization predict that COPD will become the fourth most common cause of death by 2030 and the third most common cause of chronic disability by 2020 (Lopez et al., 2006;Mathers and Loncar, 2006). Acetylcholine released by parasympathetic nerves regulates airway constriction, mucus secretion, and vasodilation through its interaction with muscarinic receptors localized in smooth muscle, mucosal glands, pulmonary vasculature, and nerve endings of the lungs (Belmonte, 2005).There are five subtypes of the muscarinic receptors, M 1 to M 5 , that are members of the superfamily of G-protein-cou-

NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively. The solution structure of NK-lysin comprises five amphipathic alpha-helices. To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin. CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of alpha-helical peptides. The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin. One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive. Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them. Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity. These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.

To study the conformational requirements of epitopes from a conserved antigenic area (area II) of respiratory syncytial (RS) virus fusion (F) glycoprotein, peptides of increasing length containing amino acids essential for these epitopes were synthesized. The synthetic peptides were tested for binding to a panel of neutralizing monoclonal antibodies (MAbs) for this area as well as to rabbit hyperimmune and human convalescent antisera. Antibody binding was dependent on peptide length; thus, a 61-residue peptide spanning amino acids 215 to 275 of the F 1 subunit (peptide F215-275) reacted with more antibodies than a shorter (41-residue) peptide F235-275, and this one with more than the (21-residue) peptide F255-275. Most human convalescent sera contained antibodies that reacted with peptides F215-275 and F235-275 but failed to react with F255-275. The results of antibody binding could be related to the structure adopted by the peptides in solution, as determined by circular dichroism spectroscopy and susceptibility of peptides to trypsin digestion. Pretreatment of peptide F215-275 with SDS abolished reactivity with certain MAbs, supporting the notion that higher order structures were needed for antibody binding. High titre anti-peptide antisera were induced in rabbits inoculated with the peptides; however, these sera failed to react with the native F molecule. In mice, only the largest F215-275 peptide induced an anti-peptide response, but their sera reacted poorly with the native F protein and the animals were not protected against an RS virus challenge. These results illustrate the potential use of synthetic peptides in studies of the F protein physical and antigenic structures as well as the problems in designing synthetic RS virus vaccines.

The Glul-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of -9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 'H-NMR at 500 MHz and values for the equilibrium association constants (K,) determined: rK1, K, -4.6 m"' ; rK2, K, -3.3 mM"; K4, KO -6.2 m"' ; K5, KO -2.3 mM". Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Ne-acetyl-L-lysine methyl ester ( K , < 0.6 mM"), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3, which is devoid of a LBS.CB-NTP and L-NTP 'H-NMR spectra were assigned and interproton distances estimated from 'H-'H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-AspGlu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys50 within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectraand exhibit fast amide 'H-2H exchange in 2H20, suggestive of high flexibility.Keywords: kringle domains; plasminogen activation peptide; plasminogen N-terminal peptide; preactivation peptide structure Reprint requests to: M. Llinas, Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213; e-mail: Ilinas+ @andrew.cmu.edu. Abbreviations; ID, one-dimensional; 2D, two-dimensional; 6-AHA, 6-aminohexanoic acid; AcArgOMe, N"-acetyl-L-arginine methyl ester; AcLysOMe, N"-acetyl-L-lysine methyl ester; Acm, acetamidomethyl group; ADC, anti-distance constraints; CB-NTP, CNBr cleaved N-terminal peptide from human plasminogen, Glul-HSer57; CD, circular dichroism; COSY, 2D chemical shift correlated spectroscopy; DIEA, N,N-diisopropylethylamine; DMF, N,Ndimethylformamide; DSS, 3-(trimethylsilyl)-l-propanesulfonic acid, sodium salt; EM, energy minimization; Et20, anhydrous diethyl ether; FmoclrBu, 9-fluorenylmethyloxycarbonyl/terf-butyl; HATU, N-[(dimethylamino) (lH-1,2,3-triazolo[4,5-b]pyridin-I-yl) methylenel-N-methyl methanaminium hexafluorophosphate N-oxide; HOAt, 7-aza-I-hydroxy-benzotriazole; HOAc, glacial a...

Synthetic peptides have been used to mimic the main antigenic site of foot-and-mouth disease virus (FMDV) of serotype C and of several variant isolates. This region includes multiple continuous B cell epitopes. The effect of single amino acid replacements, individually or in combination, on antigen specificity has been evaluated using monoclonal antibodies. Quantitative enzyme immunodot assays have shown that both additive and non-additive effects of multiple replacements occur in continuous B cell epitopes, with regard to antibody recognition. Antigenically critical single replacements may be compensated by other, non-critical replacements. Thus, the role of a single amino acid on antibody recognition depends on the sequence context in the antigenic domain. The non-additive effects of multiple replacements may modulate the extent of antigenic diversification of highly variable RNA viruses, and keep viruses confined within antigenic groups by precluding linear antigenic divergence.