Structural and functional investigation of polymyxins. Structure and biological properties of polymyxin M analogs

The effect of proteinases of plant and microbial origin on polymyxin M was studied. It was shown that this antibiotic was absolutely stable to the effect of papain and ficin. On hydrolysis with subtilisin there formed polymyxin decyclized analogs not described earlier. Their isolation, purification and biological activity are described. The structure of these compounds was assessed by one- and two-dimensional 1H NMR spectroscopy. The role of various functional groups, their space orientation and impact on antimicrobial activity of the compounds are discussed.     

Synthesis and antitumor evaluation of benzopsoralen analogues

The synthesis of five new tetracyclic benzopsoralen analogues, compounds 2-6, with 9H-xanthen-9-one or 9H-carbazole frameworks, is described. Their inhibitory effects on the growth of three human tumor cell lines (MCF-7, SF-268, and NCI-460) were evaluated, and discussed in terms of structure-activity relationship, taking into account both geometric and electronic features. Generally, the angular compounds showed significant biological activities, but the arrangement of functional groups also contributed to the overall activity.     

Scavenger and antioxidant properties of ten synthetic flavones.

To study the effect of the hydroxyl groups on biological activities of flavones, we synthesized 10 polyhydroxyflavones with varied substitution patterns. The abilities of the 10 compounds to act as radical scavengers were investigated using chemiluminescence in two biological models: the xanthine/xanthine oxidase system and the oxidative burst of rat alveolar macrophages. Stable radical formation was observed by electron spin resonance (ESR) spectroscopy. We found that the presence of the pyrogallol moiety in the B component of flavones gave rise to radical scavenger activity and that C-6 substituted hydroxyl group may also provide the basis for biological activity. Furthermore, compounds with a hydroxyl at C-7 position appeared to be xanthine oxidase inhibitors. One particular compound exhibited radical scavenger activity and xanthine oxidase inhibition. This type of compound should prove to be useful in the treatment of ischemia, for which both properties were required.     

Structural alerts for predicting clastogenic activity of pro-oxidant flavonoid compounds: quantitative structure-activity relationship study

Flavonoids have been reported to exert multiple biological effects that include acting as pro-oxidants at very high doses. The authors determined a structural alert to identify the clastogenic activity of a series of flavonoids with pro-oxidant activity. The methodology was based on a quantitative structure-activity relationship (QSAR) study. Specifically, the authors developed a virtual screening method for a clastogenic model using the topological substructural molecular design (TOPS-MODE) approach. It represents a useful platform for the automatic generation of structural alerts, based on the calculation of spectral moments of molecular bond matrices appropriately weighted, taking into account the hydrophobic, electronic, and steric molecular features. Therefore, it was possible to establish the structural criteria for maximal clastogenicity of pro-oxidant flavonoids: the presence of a 3-hydroxyl group and a 4-carbonyl group in ring C, the maximal number of hydroxyl groups in ring B, the presence of methoxyl and phenyl groups, the absence of a 2,3-double bond in ring C, and the presence of 5,7 hydroxyl groups in ring A. The presented clastogenic model may be useful for screening new pro-oxidant compounds. This alert could help in the design of new and efficient flavonoids, which could be used as bioactive compounds in nutraceuticals and functional food.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Orally active cephalosporins. Part 3: synthesis, structure-activity relationships and oral absorption of novel C-3 heteroarylmethylthio cephalosporins

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-(heteroarytmethylthio)cephalosporins was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity was markedly influenced by the structure of the heteroaromatic ring moiety. Oral absorption was influenced by the heteroaromatic ring moiety as well as by the arrangement of heteroatoms. Among these compounds, FK041 (2o), having a 4-pyrazolylmethylthio moiety, showed potent antibacterial activity against both gram-positive and gram-negative bacteria including Haemophilus influenzae. Further, it showed higher oral absorption than CFDN.     

Bacterial metabolism of quaternary ammonium compounds.

Of 10 quaternary ammonium compounds tested for biodegradation by the biological oxygen demand technique, only decyl- and hexadecyltrimethylammonium bromides were decomposed by organisms derived from sewage and soil. A mixture consisting of individual strains of Pseudomonas and Xanthomonas grew in solutions containing decyltrimethylammonium bromide as sole carbon source. The xanthomonad metabolized this quaternary ammonium compound in the presence of other organic molecules. The products of this activity included 9-carboxynomyl- and 7-carboxyheptyltrimethylammonium, suggesting that the terminal carbon of the decyl moiety is oxidized and the resulting carboxylic acid is subject to beta-oxidation.     

Optical properties of DNA complexes with antitumor compounds of bivalent platinum

The optical properties of the DNA complexes with the compounds of bivalent platinum were studied. The compounds differed by the nature of the anionic and neutral ligands and their spatial arrangement about the platinum atom. It was shown that the same as cis-[Pt (NH3)2Cl2] the platinum compounds with the biological activity, i.e. [Pt (en) Cl2], cis-[PtNH3 (Bz) Cl2] and cis-[Pt (NH3)2NO2Cl] induced at low values of r (a ratio of the number of the platinum moles added to the number of the DNA nucleotide moles in the solution) an increase in the amplitude of the positive band in the spectrum of the circular dichroism (CD) of the linear DNA and a marked decrease in the amplitude of the negative band in the spectrum of the CD of the liquid crystalline microphase of DNA formed in the presence of polyethyleneglycol. By the character of the action on the CD spectrum of the linear and condensed DNA [Pt (tetrameen)Cl2] which had no selective antimitotic effect might be referred to the above platinum compounds. Trans-[Pt (NH3)2NO2Cl], [PtNH3PyCl2], cis-[Pt (NH3)2(NO2)2] and [Pt (NH3)3Cl]Cl having no biological activity either induced only a decrease in the amplitude of the positive band in the CD spectrum of the linear DNA or had no effect on the CD spectrum. The effect of these compounds on the CD spectrum of the liquid crystalline microphase of DNA was slightly pronounced or not observed.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.     

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs.     

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.     

Conformational studies of stereoisomeric 14-membered cyclic enkephalin analogues containing 1-naphthylalanine at the fourth position: chirality effect of leucine at the fifth position on biological activity and receptor selectivity.

In order to study structure-activity relationships of enkephalin-related analogues, we report the biological activity and conformational analysis of four 14-membered cyclic enkephalin analogues with beta-(1-naphthyl) alanine in place of phenylalanine at the fourth position, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-(L and D)-Leu]. The L-beta Nal(1)-containing analogues display higher activity at both the mu and delta receptors than the corresponding analogues with the L-Phe residue. In contrast to the linear enkephalins, the cyclic analogues with the D-beta Nal(1) residue are also active at the mu receptor since the relative spatial arrangement of functional groups required for biological activity is achieved by the constrained nature of the cyclic molecules. A comparison of the findings from the conformational analysis and biological assays establishes that relatively extended structures, in which the two aromatic side chains are oriented in opposite directions with a approximately 14 A separation, is required for activity at the mu receptor. On the other hand, folded conformations with nearly parallel orientations and a close proximity (less than 10A) of the aromatic rings of the Tyr and beta Nal(1) residues are required for activity at the delta receptor. It should be noted that the overall structures and thus the biological profiles of the 14-membered cyclic enkephalin analogues are strongly dependent on the conformation of the second residue. The folded conformations with parallel orientation of the two aromatic side chains of Tyr-c[D-A2bu-Gly-L-beta Nal(1)-D-Leu] is stabilized by an interaction between the Tyr phenolic OH proton and beta Nal(1) C*O groups. This analogue, which shows the highest activity at both the mu and delta receptors among the four stereoisomers studied, displays an increase of the fraction of the side-chain chi 1 = t conformer for the beta Nal(1) residue. It is concluded that the incorporation of the D-Leu residue at the fifth position increases the relative fraction of the folded conformations with parallel orientation of the aromatic side chains, and hence enhances activity at the delta receptor as compared to the corresponding L-Leu containing analogue.     

Molecular arrangements of sphingolipids. The monolayer behaviour of ceramides

A series of synthetic ceramides have been studied at the air-water interface by recording the surface pressure-area isotherms at continuous compression.

Ceramides that contain a 4,5-trans-double bond in the long chain base were found to condense into a close-packed arrangement with vertical chains already at a very low surface pressure. The corresponding saturated compounds adopt a similar close packed arrangement only at high surface pressure. At 30 dynes/cm, a lateral pressure representative of biological membranes, the area per molecule and compressibility was further found to depend on the number and configuration of the hydroxyl groups. The presence of a 2-D-hydroxyl group in the fatty acud generally promotes the condensation. A similar effect is observed if the long chain base contains a 4-D-hydroxyl group. Cis-double bonds or methyl branches in the fatty acid chain, which increase the space requirement, limit the lateral interaction of the polar group. However the 15-cis-double bond of nervonic acid can be accommodated without any distortion of the close-packing arrangement.     

The Types and Distribution of Thiobacilli in Biological Systems Treating Carbonization Effluents

Summary: An extensive investigation of 30 strains of bacteria which oxidize inorganic sulphur compounds led to the recognition of three major groups. A study of the occurrence of these groups in biological effluent systems suggested that the organisms generally believed to be responsible for the oxidation of thiosulphate and thiocyanate, the autotrophic thiobacilli, were absent in many instances. It is suggested that in these instances heterotrophic organisms, which are found throughout all the systems, may be responsible for the destruction of the sulphur compounds. A heterotrophic organism which destroys thiocyanate, but not thiosulphate, has been isolated.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Theoretical studies on Β-lactam antibiotics VI: Conformational analysis and structure-activity relationships of penicillin sulfoxides and cephalosporins

Conformational energy calculations were carried out on penicillin α-and Β-sulfoxides and δ²- and δ³-cephalosporins, in order to identify the structural features governing their biological activity. Results on penicillin Β-sulfoxide indicated that in its favoured conformation, the orientation of the aminoacyl group was different from the one required for biological activity. Penicillin α sulfoxide, like penicillin sulfide, favoured two conformations of nearly equal energies, but separated by a much higher energy barrier. The reduced activity of the sulfoxides despite the nonplanarity of their lactam peptide indicated that the orientations of the aminoacyl and carboxyl groups might also govern biological activity. δ³-cephalosporins favoured two conformations of nearly equal energies, whereas δ²-cephalosporins favoured only one conformation. The lactam peptide was moderately nonplanÄr in the former, but nearly planar in the latter. The differences in the.preferred orientations of the carboxyl group between penicillins and cephalosporins were correlated with the resistance of cephalosporins to penicillinases.     

Comparative chemical and biological hydrolytic stability of homologous esters and isosteres

Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.     

Alterations in the antibacterial potential of Synechococcus spp. PCC7942 under the influence of UV-B radiations on skin pathogens

Marine organisms are seen as a source of novel drugs and the discovery of new pharmaceutical is increasingly in demand. Cyanobacteria are regarded as a potential target for this as antibacterial, antiviral, antifungal, algicide and cytotoxic activities have been reported in these organisms. They have been identified as a new and rich source of bioactive compounds belonging to diversified groups. Radiation in the UV-B range interferes with various metabolic reactions by generating free radicals and active oxygen species. These deleterious compounds are inactivated by antioxidants. Among them are the carotenoids and phycocyanin which protect against photodynamic action in different ways. Stress plays an important role in the production of bioactive metabolites from organisms. Synechococcus spp. PCC7942 was studied for antibacterial activity against various pathogenic bacteria resistant to a number of available antibiotics after being exposed to UV-B radiation. The antibacterial activity of Synechococcus spp. PCC7942 was studied on five potent skin pathogens. The highest antibacterial activity was seen the methanol extracts of 24 h UV-B exposed cultures of Synechococcus spp. PCC7942. It can be concluded that there was moderate antibacterial activity. Results showed stress, solvent and dose-dependent activity. This antibacterial activity might be due to the enhanced synthesis of carotenoids and phycocyanin under UV-B stress. The purpose of the present study was to relate the inhibitory effects of the cyanobacterial compounds specifically on skin pathogens with exposure to UV-B radiation as UV protecting compounds are already reported in these organisms.