A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

Comparison of apparent ileal amino acid digestibility of feed ingredients measured with broilers, layers, and roosters

The apparent ileal digestibility of amino acids in 7 feed ingredients was determined using broilers, layers, and roosters. The ingredients included 3 cereals (wheat, sorghum, and corn), 3 oilseed meals (canola, cottonseed, and soybean meals), and 1 animal protein meal (meat and bone meal). Dietary protein in the assay diets was supplied solely by the test ingredient. All diets contained 20 g/kg of acid-insoluble ash as an indigestible marker, and each diet was offered ad libitum in mash form to 5 replicate pens of broilers and layers, and 4 replicate pens of roosters. The digestibility coefficients of individual amino acids for wheat, corn, and sorghum were higher (P < 0.05) in broilers than in layers and roosters. The digestibility of most amino acids for corn and sorghum was higher (P < 0.05) in roosters compared with those in layers, whereas the digestibility for wheat in layers and roosters was similar. In general, the digestibility of amino acids in canola meal, cottonseed meal, and meat and bone meal was similar among the 3 classes of chickens. The digestibility of amino acids in soybean meal was higher (P < 0.05) for layers compared with those for broilers and roosters but similar between broilers and roosters. These results suggest that the class of chickens significantly influenced the apparent ileal digestibility of amino acids in some feed ingredients.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Spectroscopic characterization of copper(II) binding to the immunosuppressive drug mycophenolic acid

Mycophenolic acid (MPA) is a drug that has found widespread use as an immunosuppressive agent which limits rejection of transplanted organs. Optimal use of this drug is hampered by gastrointestinal side effects which can range in severity. One mechanism by which MPA causes gastropathy may involve a direct interaction between the drug and gastric phospholipids. To combat this interaction we have investigated the potential of MPA to coordinate Cu(II), a metal which has been used to inhibit gastropathy associated with use of the NSAID indomethacin. Using a range of spectroscopic techniques we show that Cu(II) is coordinated to two MPA molecules via carboxylates and, at low pH, water ligands. The copper complex formed is stable in solution as assessed by mass spectrometry and H-1 NMR diffusion experiments. Competition studies with glycine and albumin indicate that the copper-MPA complex will release Cu(II) to amino acids and proteins thereby allowing free MPA to be transported to its site of action. Transfer to serum albumin proceeds via a Cu(MPA)(albumin) ternary complex. These results raise the possibility that copper complexes of MPA may be useful in a therapeutic situation.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Antioxidants and protein oxidation

Proteins are susceptible to oxidation by reactive oxygen species, where the type of damage induced is characteristic of the denaturing species. The induction of protein carbonyls is a widely applied biomarker, arising from primary oxidative insult. However, when applied to complex biological and pathological conditions it can be subject to interference from lipid, carbohydrate and DNA oxidation products. More recently, interest has focused on the analysis of specific protein bound oxidised amino acids. Of the 22 amino acids, aromatic and sulphydryl containing residues have been regarded as being particularly susceptible to oxidative modification, with L-DOPA from tyrosine, ortho-tyrosine from phenylalanine; sulphoxides and disulphides from methionine and cysteine respectively; and kynurenines from tryptophan. Latterly, the identification of valine and leucine hydroxides, reduced from hydroperoxide intermediates, has been described and applied. In order to examine the nature of oxidative damage and protective efficacy of antioxidants the markers must be thoroughly evaluated for dosimetry in vitro following damage by specific radical species. Antioxidant protection against formation of the biomarker should be demonstrated in vitro. Quantification of biomarkers in proteins from normal subjects should be within the limits of detection of any analytical procedure. Further to this, the techniques for isolation and hydrolysis of specific proteins should demonstrate that in vitro oxidation is minimised. There is a need for the development of standards for quality assurance material to standardise procedures between laboratories. At present, antioxidant effects on protein oxidation in vivo are limited to animal studies, where dietary antioxidants have been reported to reduce dityrosine formation during rat exercise training. Two studies on humans have been reported last year. The further application of these methods to human studies is indicated, where the quality of the determinations will be enhanced through inter-laboratory validation.

Hallmarks of protein oxidative damage in neurodegenerative diseases:Focus on Alzheimer's disease

The pathogenesis of several neurodegenerative diseases, including Alzheimer's disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer's disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer's disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process. © 2007 Springer-Verlag.

Novel formulation strategies for the fabrication of lyophilised orally disintegrating tablets

Orally disintegrating Tablets (ODTs), also known as fast-disintegrating, fast-melt or fast-dissolving tablets, are a relatively novel dosage technology that involves the rapid disintegration or dissolution of the dosage form into a solution or suspension in the mouth without the need for water. The solution containing the active ingredients is swallowed, and the active ingredients are then absorbed through the gastrointestinal epithelium to reach the target and produce the desired effect. Formulation of ODTs was originally developed to address swallowing difficulties of conventional solid oral dosage forms (tablets and capsules) experienced by wide range of patient population, especially children and elderly. The current work investigates the formulation and development of ODTs prepared by freeze drying. Initial studies focused on formulation parameters that influence the manufacturing process and performance of lyophilised tablets based on excipients used in commercial products (gelatin and saccharides). The second phase of the work was followed up by comprehensive studies to address the essential need to create saccharide free ODTs using naturally accruing amino acids individually or in combinations. Furthermore, a factorial design study was carried out to investigate the feasibility of delivering multiparticulate systems of challenging drugs using a novel formulation that exploited the electrostatic associative interaction between gelatin and carrageenan. Finally, studies aimed to replace gelatin with ethically and morally accepted components to the end users were performed and the selected binder was used in factorial design studies to investigate and optimise ODT formulations that incorporated drugs with varies physicochemical properties. Our results show that formulation of elegant lyophilised ODTs with instant disintegration and adequate mechanical strength requires carful optimisation of gelatin concentration and bloom strength in addition to saccharide type and concentration. Successful formulation of saccharides free lyophilised ODTs requires amino acids that crystallise in the frozen state or display relatively high Tg', interact and integrate completely with the binder and, also, display short wetting time with the disintegrating medium. The use of an optimised mixture of gelatin, carrageenan and alanine was able to create viscous solutions to suspend multiparticulate systems and at the same time provide tablets with short disintegration times and adequate mechanical properties. On the other hand, gum arabic showed an outstanding potential for use as a binder in the formulation of lyophilised ODTs. Compared to gelatin formulations, the use of gum arabic simplified the formulation stages, shortened the freeze drying cycles and produced tablets with superior performance in terms of the disintegration time and mechanical strength. Furthermore, formulation of lyophilised ODTs based on gum arabic showed capability to deliver diverse range of drugs with advantages over commercial products.

Amino acid-modified spray-dried powders with enhanced aerosolisation properties for pulmonary drug delivery

In this study, the amino acids arginine, aspartic acid, leucine, phenylalanine and threonine were investigated as 'dispersibility enhancers' in spray-dried powders for inhalation. Parameters such as spray-dried yield, tapped density, and Carr's Index were not predictive of aerosolisation performance. In addition, whilst the majority of amino acid-modified powders displayed suitable particle size distribution for pulmonary administration and potentially favourable low moisture content, in vitro particle deposition was only enhanced for the leucine-modified powder. In summary, leucine can be used to enhance the dispersibility and aerosolisation properties of spray-dried powders for pulmonary drug delivery. © 2007 Elsevier B.V. All rights reserved.

Proteolytic activity amongst selected Saprolegnia species

Saprolegia ssp. effectively utilized the protein casein as a sole source of carbon, nitrogen and sulphur, indicating considerable proteolytic activity. In the presence of a more simple carbon source such as glucose, which was readily assimilated, catabolite repression was not observed and casein exploitation was enhanced. Free proteinase activity was not detected by a number of methods, irrespective of culture conditions. However, clearing by mycelia of skimmed milk agar or agar amended with bacteria demonstrated a close association between proteinases and hyphae, suggestive of natural immobilization of proteinases. Casein breakdown was accompanied by release of individual amino acids and ammonia. The latter, indicative of amino acid assimilation and metabolism, was also associated with an increase in pH of culture medium. Single amino acids did not support growth of Saprolegnia but in combination with other amino acids, methionine encouraged greatest biomass production. Certain groupings of amino acids affected growth in a manner which departed from that expected, as assessed by multifactorial analysis of variance, and either enhanced or reduced growth.

Functional polymers for biomedical application : synthesis and applications

Aromatic and aliphatic diacid chlorides were used to condense naturally occurring diamino acids and their esterified derivatives. It was anticipated the resulting functional polyamides would biodegrade to physiologically acceptable compounds and show pH dependant solubility could be used for biomedical applications ranging from enteric coatings to hydrosoluble drug delivery vehicles capable of targeting areas of low physiological pH. With these applications in mind the polymers were characterised by infra red spectroscopy, gel permeation chromatography and in the case of aqueous soluble polymers by potentiometric titration. Thin films of poly (lysine ethyl ester isophthalamide) plasticised with poly (caprolactone) were cast from DMSO/chloroform solutions and their mechanical properties measured on a Hounsfield Hti tensiometer. Interfacial synthesis was investigated as a synthetic route for the production of linear functional polyamides. High molecular weight polymer was obtained only when esterified diamino acids were condensed with aromatic diacid chlorides. The method was unsuitable for the production of copolymers of free and esterified amino acids with a diacid chloride. A novel miscible mixed solvent single phase reaction was investigated for production of copolymers of esterified and non-esterified amino acids with diacid chlorides. Aliphatic diacid chlorides were unsuitable for condensing diamino acids using this technique because of high rates of hydrolysis. The technique gave high molecular weight homopolymers from esterified diamino acids and aromatic diacid chlorides.