We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

This paper reports on a novel continuous-flow hydrogenation reactor and its integration with a liquid handler to generate a fully automated high-throughput hydrogenation system for library synthesis. The reactor, named the H-Cube, combines endogenous hydrogen generation from the electrolysis of water with a continuous flow-through system. The system makes significant advances over current batch hydrogenation reactors in terms of safety, reaction validation efficiency, and rates of reaction. The hydrogenation process is described along with a detailed description of the device's main parts. The reduction of a series of functional groups, varying in difficulty up to 70 degrees C and 70 bar are also described. The paper concludes with the integration of the device into an automated liquid handler followed by the reduction of a nitro compound in a high throughput manner. The system is fully automated and can conduct 5 reactions in the time it takes to perform and workup one reaction manually on a standard batch reactor.

Insects respond to a bacterial challenge by rapidly synthesizing a diverse range of antibacterial and antifungal peptides. One of them, drosocin, a 19-residue proline-rich antibacterial peptide, was isolated from Drosophila. This peptide carries a disaccharide moiety attached to a threonine residue in mid-chain position. The present report describes the enlarged-scale chemical synthesis of drosocin, glycosylated with Gal(~l+3)GalNAc(al+O). We have studied the range of activity of the synthetic glycopeptide, of two truncated glycosylated isoforms, and of the unglycosylated L and D enantiomers. Both isolated and chemically synthesized drosocins carrying the disaccharide display the same antibacterial activity. Using circular dichroic spectroscopy we demonstrated that the 0-linked disaccharidic motif did not affect the backbone conformation of drosocin. The antibacterial activity of the synthetic glycopeptide was directed against gram-negative strains with the exception of the gram-positive bacteria Micrococcus luteus. Deletion of the first five N-terminal residues completely abolished the activity of drosocin. As a first approach to the study of the mode of action of drosocin, we have synthesized a non-glycosylated D enantiomer and, using this molecule, we have shown that drosocin may act on the gram-negative bacteria through a stereospecific target.

No abstract

No abstract

No abstract

The determination of peptide stability in human serum (HS) or plasma constitutes a powerful screening assay for eliminating unstable peptides from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selected autoimmune disorders such as rheumatoid arthritis and insulin-dependent diabetes. For most of the l-amino acid peptides studied, the predominant degradation mechanism is exopeptidase-catalyzed cleavage. Peptides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substitutions. In addition, N-acetylglucosamine glycopeptides were significantly stabilized, even when the glycosylation site was several amino acids from the predominant site(s) of cleavage. This indicates that long-range stabilization is possible, and likely due to altered peptide conformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala peptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.

The high attrition rate of drug candidates during clinical trials for poor pharmacokinetic and metabolic properties has created a need to do these studies as early as it is possible during the drug discovery process. In addition the most successful drug is often not the most potent one but the one that has the suitable level of potency, safety, and pharmacokinetics. Science and technology development during the last few years and the generation of last databases and information has created the basis for doing early experimental PK and ADME studies in addition to eADME. Similarly, testing safety features as early as possible is key to affordable drug discovery and development. Throughput and cost are crucial for early application. In silico methods have by far the highest throughput, followed by the in vitro and in vivo approaches. On the other hand, with regard to relevance and reliability of data the ranking is the opposite. The great challenge for in silico methods is generation of models that correlate more closely with in vivo systems. For the in vitro assays increasing the throughput is an absolute must. Ex silico methods that combine in silico predictions with experimental methods are new additions to the scientific repertoire (e.g. Chromatographic Hydrophobicity Index that is deduced from the reverse phase HPLC data can be used for calculation of lipophilicity). The emerging new approaches have clear impact on the design of early stage screening and combinatorial libraries. In addition to the Lipinski's rules descriptors such as number of rotatable bonds, number of aromatic rings, branching behavior and polar surface area (PSA) are commonly used is the drug design process.