Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.     

Reconstruction of pathways associated with amino acid metabolism in human mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochondria. Human mitochondrial proteins determined by experimental and computational methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mitochondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which predicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we reconstructed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.     

Phospholipase D1 regulates phagocyte adhesion

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation, transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Because phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen, or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy indicated that PLD1 exhibited partial colocalization with actin filaments at the adherent interface, in proximity to the focal adhesion protein, paxillin. Reductions in PLD activity by chemical inhibitors or specific short-interfering RNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion and was accompanied by reductions in total cellular F-actin. These data support the hypotheses that adhesion stimulates PLD activity, and that PLD1 regulates the initial stages of phagocyte adhesion. Stimulation of PLD activity may promote adhesion-dependent phagocyte effector responses.     

FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F. graminearum

Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.     

Locally defined protein phylogenetic profiles reveal previously missed protein interactions and functional relationships

Phylogenetic profiles encode patterns of presence or absence of genes across genomes, and these profiles can be used to assign functional relationships to nonhomologous pairs of proteins (Pellegrini et al., Proc Natl Acad Sci USA 1999;96:4284-4288). Although it is well known that many proteins were created from combinations of domains, most of the existing implementations of phylogenetic profiles do not consider this fact. Here, we introduce an extension that considers the multidomain nature of proteins and test the method against the known interaction data sets. Whereas earlier implementations associated one entire sequence with one protein phylogenetic profile (Single-Profile), our method instead breaks the sequence into a set of segments of predetermined size and constructs a separate profile for each segment (Multiple-Profile). The results show that the Multiple-Profile method performs as well as the Single-Profile method. However, the two methods share, surprisingly, a small fraction of their predictions, indicating that the Multiple-Profile method can detect known interactions missed by the Single-Profile method. Thus, the Multiple-Profile method can be used with other methods to determine functional relationships on a genome scale with wider coverage.     

Discovery of N-{N-[(3-cyanobenzene) sulfonyl]-4(R)-(3,3-difluoropiperidin-1-yl)-(l)-prolyl}-4-[(3′,5′-dichloro-isonicotinoyl) amino]-(l)-phenylalanine (MK-0617), a highly potent and orally active VLA-4 antagonist

A series of prolyl-N-isonicotinoyl-(L)-4-aminophenylalanine derivatives substituted at the proline 4-position with cyclic amines was evaluated as VLA-4 antagonists. The ring size and presence or absence of fluorine affected potency and receptor occupancy. The analog with 3,3-difluoropiperidine at the proline 4-position (13) was the most potent compound and had very good duration of receptor occupancy in vitro. The ethyl ester prodrug of 13 demonstrated excellent receptor occupancy after oral dosing in rats.     

Binuclear copper and zinc complexes based on polypyridyl ligand 2,3,5,6-tetra(2-pyridyl)pyrazine (tppz): Synthesis, spectral and structural characterization

The reaction of MCl₂ · 2H₂O (M = Cu, Zn) with 2,3,5,6-tetra(2-pyridyl)pyrazine (tppz) (referred hereafter as L) in 2:1 molar ratio in acetonitrile at room temperature afforded binuclear complexes [M₂(κ³-L)Cl₄] [Cu (1), Zn (2)] where the ligand is bis-tridentate manner. The complexes have been characterized by elemental analyses, FAB-MS, IR, EPR, NMR and electronic spectral studies. Solid state structures of both the [Cu₂(κ³-L)Cl₄] · 5H₂O (1), [Zn₂(κ³-L)Cl₄] · H₂O (2) have been determined by single crystal X-ray analyses. A well-resolved uudd cyclic water tetramer and water monomer were reported in the crystal host of [Cu₂(κ³-L)Cl₄] · 5H₂O (1) and [Zn₂(κ³-L)Cl₄] · H₂O (2) showing the contribution of the water cluster to the stability of the crystal host 1 and 2.     

Study on the diversity of endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro

Endophytic populations were isolated from 2400 segments of Oryza sativa collected from Bhadra River Project Area, Southern India during December 2005 (Winter) and April 2006 (Summer). Overall colonization rates from surface sterilized tissues were 40.3% in roots and 25.83% in leaves during winter season, 20.15% in roots and 8.66% in leaves during summer season. Nineteen different fungal taxa, a Streptomyces sp. and bacterial species were isolated. Streptomyces sp., Chaetomium globosum, Penicillium chrysogenum, Fusarium oxysporum and Cladosporium cladosporioides were dominant endophytes in this study. Frequency of colonization between the sites, seasons and rice varieties were found to differ significantly. Dual culture studies revealed that C. globosum, P. chrysogenum and Streptomyces sp. are suitable candidates for extraction of biologically active compounds. Rice harbors many endophytic organisms and some of them have antagonistic properties against fungal pathogens.