Chemistry of biologically active benzoxazinoids

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and its desmethoxy derivative (DIBOA) are major allelochemicals produced by corn, wheat, rye and related monocotyledons. These benzoxazinone derivatives have a wide variety of biological actions, including antifungal and mutagenic activities. Structure-activity relationships of these compounds and their derivatives (benzoxazinoids), the reactivity of benzoxazinoids with nucleophiles, and the substituent effects of the 7-methoxy and 2-hydroxy groups are discussed in relation to the molecular mechanisms of the biological activities.     

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

Chemoenzymatic synthesis of chiral biologically active heterocycles

During 1995, among 1105 HIV patients explored in our department, 64 presented a deep fungic infection (5.8%). The yeast was searched for in cerebrospinal fluid, blood, urine, and bronchoalveolar aspiration. Isolated germs were Cryptococcus neoformans (95%), Candida tropicalis (1 case), Saccharomyces cerevisiae (1 case) et Aspergillus fumigatus (1 case). Results of treatment with amphotericin B were: recovery (9%), clinical success (11%), out of sight (14%), letality (66%), relapse (23%) and side effects (19%). We emphasized diagnostical and therapeutical difficulties, and bad prognostic of mycoses in patients with AIDS.     

Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules

Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.     

Visoltricin, a novel biologically active compound produced by Fusarium tricinctum

The major compound responsible for toxicity to Artemia salina of some Fusarium tricinctum strains has been isolated, and its structure has been elucidated by spectroscopical methods, i.e. UV, IR, MS, 1H-NMR and 13C-NMR. The novel compound, trivially named visoltricin, is the first imidazole derivative produced by Fusarium spp., and its structure has been established as the methyl ester of 3-[1-methyl-4-(3-methyl-2-butenyl)-imidazol-5yl]-2-propenoic acid (molecular formula C13H18N2O2; MW = 234.297). Visoltricin was toxic to A. salina larvae (LD50 = 8.5 x 10(-7) M), and inhibited the growth of six human tumour cell lines (out of 60 lines tested) at concentrations lower than 10(-5) M. Tested on rabbit eye it showed an interesting miotic activity similar to that of pilocarpine, a miotic agent largely used in the therapy of glaucoma. This biological activity could be explained in part by the anticholinesterase properties shown by visoltricin towards both human serum and pure enzymes (EC 3.1.1.7 and EC 3.1.1.8). Kinetics studies showed for visoltricin a mixed-type and reversible inhibition of the EC 3.1.1.7 enzyme with the competitive inhibition constant (Ki) = 1.9 x 10(-4) M.     

In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.     

Introducing a C-interglycosidic bond in a biologically active pentasaccharide hardly affects its biological properties

We describe here the synthesis and the biological activity of a 'C-pentasaccharide', a new analogue of the antithrombin III (AT III) binding region of heparin containing a methylene bridge in place of an interglycosidic oxygen atom. The affinity for AT III and the anti-factor Xa activity of this compound have been compared with that of the corresponding selected 'O-pentasaccharide'. Such a structural modification slightly decreased the affinity of this compound for AT III as well as its anti-factor Xa activity (880 +/- 40 anti-Xa units versus 1180 +/- 30 anti-Xa units for the C-pentasaccharide and the O-pentasaccharide, respectively). This compound therefore represents the first example of a new class of anti-factor Xa pentasaccharides containing a C-interglycosidic bond.     

Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase

BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.     

Purification of biologically active SPARC expressed in Saccharomyces cerevisiae

SPARC (secreted protein, acidic and rich in cysteine) is a secreted, Ca+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC have been mammalian bone, platelets, a basement membrane tumor, and cultured cells; most if not all preparations, however, contain platelet-derived growth factor and one or more serum proteins that bind specifically to purified SPARC. To avoid these contaminants, as well as the toxic lipid moiety associated with endotoxin, we expressed recombinant wild-type and a mutated murine SPARC in two strains of Saccharomyces cerevisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recombinant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimized for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution of Asn98 with Asp98 in the wild-type sequence. Purification of SPARC was achieved by copper-affinity and hydrophobic-interaction chromatography. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endothelial cells: inhibition of cell spreading/disruption of actin microfilaments and competition for the binding of nonrecombinant 125I-labeled SPARC to the cell surface. The availability of biologically active, recombinant SPARC will facilitate investigation of the structural and functional properties of this protein, which is expressed at high levels in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue.     

Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.     

Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.     

Improved method for the purification of biologically active transcobalamin II

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.     

Application of Kohonen Neural Networks in classification of biologically active compounds

Automated data classification is an indispensable tool in Drug Design. It allows to select homogeneous training sets or to distinguish compounds with required biological properties. The Kohonen Neural Networks (KNN) suggest new means for classification of biologically interesting compounds. In this paper, first, capabilities of KNN in data dimensionality reduction are presented as compared with the capabilities of Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). The advantages of KNN become evident with increasing data dimensionality and size of the training set. Then, new methods are suggested to evaluate the quality of KNN models. Finally, a case study on chemical and biological data is presented. The database studied includes more than 2000 organophosphorous potent pesticides. The Kohonen maps were obtained which allow to distinguish compounds with different biological behavior.     

Identification of biologically active sites in laminin an extracellular matrix protein

Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin alpha 1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.     

Modern directions in studying biologically active substances from basidial fungi

Nonconventional uses of basidial fungi as producers of valuable biologically active compounds are reviewed. Basidiomycetes are now most extensively used in the medical industry to obtain antibiotics, enzymes, lipids, and various drugs acting on the immune system (hypocholesterolemic, antiviral, hypotensive, hypoglycemic, etc.). Recent data on toxic and psychotropic substances of Basidiomycetes and of their aromatic compounds responsible for the so-called fungal flavor are also considered. Finally, the increasing significance of Basidiomycetes for human practice is underlined.     

Changes in biologically active ultraviolet radiation reaching the Earth's surface

Stratospheric ozone levels are near their lowest point since measurements began, so current ultraviolet-B (UV-B) radiation levels are thought to be close to their maximum. Total stratospheric content of ozone-depleting substances is expected to reach a maximum before the year 2000. All other things being equal, the current ozone losses and related UV-B increases should be close to their maximum. Increases in surface erythemal (sunburning) UV radiation relative to the values in the 1970s are estimated to be: about 7% at Northern Hemisphere mid-latitudes in winter/spring; about 4% at Northern Hemisphere mid-latitudes in summer/fall; about 6% at Southern Hemisphere mid-latitudes on a year-round basis; about 130% in the Antarctic in spring; and about 22% in the Arctic in spring. Reductions in atmospheric ozone are expected to result in higher amounts of UV-B radiation reaching the Earth's surface. The expected correlation between increases in surface UV-B radiation and decreases in overhead ozone has been further demonstrated and quantified by ground-based instruments under a wide range of conditions. Improved measurements of UV-B radiation are now providing better geographical and temporal coverage. Surface UV-B radiation levels are highly variable because of cloud cover, and also because of local effects including pollutants and surface reflections. These factors usually decrease atmospheric transmission and therefore the surface irradiances at UV-B as well as other wavelengths. Occasional cloud-induced increases have also been reported. With a few exceptions, the direct detection of UV-B trends at low- and mid-latitudes remains problematic due to this high natural variability, the relatively small ozone changes, and the practical difficulties of maintaining long-term stability in networks of UV-measuring instruments. Few reliable UV-B radiation measurements are available from pre-ozone-depletion days. Satellite-based observations of atmospheric ozone and clouds are being used, together with models of atmospheric transmission, to provide global coverage and long-term estimates of surface UV-B radiation. Estimates of long-term (1979-1992) trends in zonally averaged UV irradiances that include cloud effects are nearly identical to those for clear-sky estimates, providing evidence that clouds have not influenced the UV-B trends. However, the limitations of satellite-derived UV estimates should be recognized. To assess uncertainties inherent in this approach, additional validations involving comparisons with ground-based observations are required. Direct comparisons of ground-based UV-B radiation measurements between a few mid-latitude sites in the Northern and Southern Hemispheres have shown larger differences than those estimated using satellite data. Ground-based measurements show that summertime erythemal UV irradiances in the Southern Hemisphere exceed those at comparable latitudes of the Northern Hemisphere by up to 40%, whereas corresponding satellite-based estimates yield only 10-15% differences. Atmospheric pollution may be a factor in this discrepancy between ground-based measurements and satellite-derived estimates. UV-B measurements at more sites are required to determine whether the larger observed differences are globally representative. High levels of UV-B radiation continue to be observed in Antarctica during the recurrent spring-time ozone hole. For example, during ozone-hole episodes, measured biologically damaging radiation at Palmer Station, Antarctica (64 degrees S) has been found to approach and occasionally even exceed maximum summer values at San Diego, CA, USA (32 degrees N). Long-term predictions of future UV-B levels are difficult and uncertain. Nevertheless, current best estimates suggest that a slow recovery to pre-ozone depletion levels may be expected during the next half-century. (ABSTRACT TRUNCATED)