Structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase)

A series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor (1, OM99-2 with eight residues) bound to memapsin 2. Ring size and substituent effects have been investigated. Cycloamide-urethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against human brain memapsin 2 (beta-secretase).     

Potent memapsin 2 (beta-secretase) inhibitors: design, synthesis, protein-ligand X-ray structure, and in vivo evaluation

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic beta-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K(i) 1.8 nM) and cellular (IC(50)=1 nM in Chinese hamster ovary cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma A beta) in transgenic mice. The X-ray structure of protein-ligand complex of memapsin 2 revealed critical interactions in the memapsin 2 active site.     

A putative nuclear growth factor‐like globular nematode specific protein

Expressed sequence tags (ESTs) are an effective approach for discovery of novel genes. In the current study, approximately 250 ESTs of the cattle parasitic nematode Setaria digitata were examined and a cDNA clone identified whose coding sequence could not be functionally annotated by searching over publicly available genome, protein, EST and STS databases. Here, we report the extensive characterization of this ORF (UP) and its homologues using a bioinformatic approach. Uncharacterized protein (SDUP) of S. digitata consists of 204 amino acids with a predicted molecular weight and isoelectric point of 22.8KDa and 9.94, respectively. A search carried out using SDUP over nucleotide, EST and protein databases at NCBI, NEMBASE3 and Parasite Genome Database (PGD) identified homologous counterparts from the human parasitic nematodes Wuchereria bancrofti (WB), Brugia malayi (BM), Onchocerca volvulus (OV), the mouse filarial worm Litomosoides sigmodontis (LS), swine parasitic nematodes Ascaris suum (AS) and diverged counterparts from the plant parasitic nematode Meloidogyne hapla (MH) and free living nematodes Caenorhabditis elegans (CE) and Caenorhabditis briggsae (CB). Phylogenetic analyses revealed the UPs to be undergoing divergent evolution. A search of the ESTs at PGD showed that UP is expressed in all the stages of BM. Secondary structure analyses of multiply-aligned sequences of homologues using Jpred server indicated UPs to be rich in beta-pleated structures. TMMHH server and beta barrel finder programme indicated, UPs to be neither transmembrane or beta barrels proteins but are likely to be globular proteins. Further, the Motif discovery tool of MEME identified three novel potential motifs for UPS, of which only two are present in CE, CB & MH. Analyses of UPs using Signal IP, TargetP, Psort servers predicted this group of proteins to be devoid of signal peptide cleavage sites, are not mitochondrial targeting peptides but appear to be localized to the nucleus, respectively. Further analyses of the UPs using ScanProsite server for phosphorylation revealed potential sites for cAMP­ and cGMP­dependent protein kinase, Protein kinase C and Casein kinase II. Putative functional analysis using ProtFun 2.1 Server indicated UPs to be nonenzymatic, growth factor like protein. Finally, collating all the information derived from bioinformatic analyses, we conclude that the UPs of nematodes are most likely to be expressed at all stages in the life cycle, localized to the nucleus, regulated by phosphorylation, rich in beta­pleated strands and are growth factor like nematode specific proteins.     

In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors

We have previously reported structure-based design of memapsin 2 (beta-secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a "carrier peptide" penetrated the plasma membrane in cultured cells and inhibited the production of beta-amyloid (Abeta). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Abeta level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Abeta reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.     

Conversion of an amplified fragment length polymorphism marker into a co-dominant marker in the mapping of the<Emphasis Type="Italic"> Rph15</Emphasis> gene conferring resistance to barley leaf rust,<Emphasis Type="Italic"> Puccinia hordei</Emphasis> Otth.

Leaf rust, caused by Puccinia hordei, is an important disease afflicting barley (Hordeum vulgare) in many production regions of the world. The leaf rust resistance gene Rph15 was identified in an accession of wild barley (Hordeum vulgare subsp. spontaneum) and is one of the most broadly effective resistance genes known. Using amplified fragment length polymorphism (AFLP) and simple sequence repeat markers, Rph15 was mapped to chromosome 2HS in an F₂ population derived from a cross between Bowman (Rph15), a Bowman backcross-derived line carrying Rph15, and the susceptible cultivar Bowman. AFLP marker P13M40 co-segregated with Rph15 in this mapping population and two others involving Bowman (Rph15) and cultivars Proctor and Nudinka. The dominant AFLP marker P13M40 was converted to a co-dominant PCR-based marker that may be useful in breeding programs employing marker-assisted selection. The allelic relationship between Rph15 and the gene Rph16, also mapping to chromosome 2HS, was studied. The lack of segregation in F₂ progeny derived from the two resistance sources indicates that Rph15 and Rph16 are alleles of the same locus.Communicated by F. Salamini     

Genetic differentiation among Sri Lankan traditional rice (Oryza sativa) varieties and wild rice species by AFLP markers

Traditional rice varieties are one important component of the biodiversity of Sri Lanka. However, no proper studies have been performed on genetic diversity of the Sri Lankan traditional rice varieties used in our breeding programs. In the present study, the genetic diversity of 46 traditional rice (Oryza sativa) varieties and 5 wild rice species is assesed using Amplified Fragment Length Polymorphism (AFLP) markers. Ten primer combinations generated a total of 784 fragments. Of these, 772 fragments were polymorphic (98.4%). UPGMA analysis based on Jaccard's similarity separated the accessions into four major clusters. A cophenetic correlation with r=0.786 strongly supported the clustering pattern of UPGMA dendrogram. A principal coordinate analysis (PCoA) also confirmed the UPGMA clusters. Accessions referred to the same cluster showed similar morphological characteristics (e.g. height, grain colour etc.) while accessions which are known to be morphologically distinct appeared genetically separated. In addition, the clustering pattern distinctly separated lowland and upland rice varieties. This genetic diversity assessment at the molecular level provides reliable information for selection of germplasm in the development of new rice varieties and in conservation of traditional rice genetic resources.