Copper-mediated nuclease activity of jadomycin B.

The natural product jadomycin B, isolated from Streptomyces venezeulae ISP5230, has been found to cleave DNA in the presence of Cu(II) ions without the requirement for an external reducing agent. The efficiency of DNA cleavage was probed using supercoiled plasmid DNA in buffered solution as a model environment. EC50 and t(½) values for cleavage were 1.7 µM and 0.75 h, respectively, and varied ± 5% with the particular batch of plasmid and jadomycin employed. While UV-vis spectroscopy indicates that the cleavage event does not involve direct binding of jadomycin B to DNA, a stoichiometric Cu(II) preference for optimum cleavage suggests a weak binding interaction between jadomycin B and Cu(II) in the presence of DNA. The Cu(II)-mediated cleavage is greatly enhanced by UV light, which implicates the jadomycin B radical cation and Cu(I) as potential intermediates in DNA cleavage. Evidence in favor of this hypothesis was derived from a mechanistic assay which showed reduced cleavage as a function of added catalase and EDTA, scavengers of H2O2 and Cu(II), respectively. Thus, jadomycin B may serve as a source of electrons for Cu(II) reduction, producing Cu(I) which reacts with H2O2 to form hydroxyl radicals that cause DNA strand scission. In addition, scavengers of hydroxyl radicals and superoxide also display inhibitory effects, underscoring the ability of jadomycin B to produce a powerful arsenal of deleterious oxygen species when copper is present.

Isolation and characterisation of amphotericin B analogues and truncated polyketide intermediates produced by genetic engineering of Streptomyces nodosus.

Amphotericin B is a powerful but toxic drug used against fungal infections and leishmaniases. These diseases would be treated more effectively if non-toxic amphotericin derivatives could be produced on a large scale at low cost. Genetic manipulation of the amphotericin B producer, Streptomyces nodosus, has previously led to the detection and partial characterisation of 8-deoxyamphotericin B, 16-descarboxyl-16-methyl-amphotericin B, 15-deoxy-16-descarboxyl-16-methyl-15-oxo-amphotericin B, 7-oxo-amphotericin B and pentaene analogues. Here we report improved production and purification protocols that have allowed detailed chemical analyses of these compounds. The polyketide synthase product 8-deoxy-16-descarboxyl-16-methyl-amphoteronolide B was identified for the first time. In addition, the ketoreductase 10 domain of the polyketide synthase was specifically inactivated by targeted gene replacement. The resulting mutants produced truncated polyketide intermediates as linear polyenyl-pyrones.

Diverse DNA-cleaving capacities of the jadomycins through precursor-directed biosynthesis.

Gel mobility assays were used to establish that some members of the jadomycin family of natural products act as DNA cleaving agents. Moreover, it was found that subtle structural changes generated through the use of precursor-directed biosynthesis lead to marked effects on the DNA-damaging properties of these glycosylated polyketide-derived natural products.

Engineered synthesis of 7-oxo- and 15-deoxy-15-oxo-amphotericins: insights into structure-activity relationships in polyene antibiotics.

Site-directed mutagenesis and gene replacement were used to inactivate two ketoreductase (KR) domains within the amphotericin polyketide synthase in Streptomyces nodosus. The KR12 domain was inactivated in the DeltaamphNM strain, which produces 16-descarboxyl-16-methyl-amphotericins. The resulting mutant produced low levels of the expected 15-deoxy-15-oxo analogs that retained antifungal activity. These compounds can be useful for further chemical modification. Inactivation of the KR16 domain in the wild-type strain led to production of 7-oxo-amphotericin A and 7-oxo-amphotericin B in good yield. 7-oxo-amphotericin B was isolated, purified, and characterized as the N-acetyl methyl ester derivative. 7-oxo-amphotericin B had good antifungal activity and was less hemolytic than amphotericin B. These results indicate that modification at the C-7 position can improve the therapeutic index of amphotericin B.

Stereochemical integrity of oxazolone ring-containing jadomycins.

The jadomycins are a series of natural products produced by Streptomyces venzuelae ISP5230 in response to ethanol shock. A unique structural feature of these angucyclines is the oxazolone ring, the formation of which is catalyzed by condensation of a biosynthetic aldehyde intermediate and an amino acid. The feeding of enantiomeric forms of alpha-amino acids indicates that the amino acid is incorporated by S. venezuelae ISP5230 without isomerization at the alpha-carbon. The characterization of the first two six-membered E-ring-containing jadomycins is reported. These precursor-directed biosynthesis studies indicate flexibility in the acceptor substrate specificity of the glycosyltransferase, JadS. Analysis of cytotoxicity data against two human breast cancer cell lines indicates that the nature of the substitution at the alpha-carbon, rather than the stereochemistry, influences biological activity.

Substrate flexibility of a 2,6-dideoxyglycosyltransferase.

We report the first 2,6-dideoxysugar-O-glycosyltransferase with substrate flexibility at the 2 position, confirm the function of a putative NDP-hexose 2,3-dehydratase in the jadomycin B biosynthetic gene cluster and deduce the substrate flexibility of downstream enzymes in l-digitoxose assembly, enabling reprogramming of biosynthetic gene clusters to modify sugar substituents.

Ring-opening dynamics of jadomycin A and B and dalomycin T.

[structure: see text] A novel oxazolone ring-opening and interconversion process between the two jadomycin diastereomeric forms has been characterized by NMR spectroscopy. An analogue, dalomycin T, has been isolated for the first time and does not undergo interconversion.

Adenophostin A and analogues modified at the adenine moiety: synthesis, conformational analysis and biological activity.

The synthesis of adenophostin A (2) and two analogues [etheno adenophostin (4) and 8-bromo adenophostin (5)] modified at the adenine moiety, is reported. A combination of NMR analysis and molecular modelling was used to compare their structures in solution and determined that they all adopt very similar conformations. The analogues were tested for their ability to mobilise Ca(2+) from DT40 cells expressing recombinant Type 1 rat Ins(1,4,5)P(3)R which reveals etheno adenophostin as a high affinity fluorescent probe of the Ins(1,4,5)P(3)R. 8-Bromo adenophostin was only slightly less potent. The biological results support our current hypothesis regarding the binding mode of adenophostin A at the Ins(1,4,5)P(3)R, i. e. that a cation-pi interaction between the base moiety and Arg 504 of the receptor in combination with H-bonding may be responsible for the high potency of adenophostin A relative to Ins(1,4,5)P(3).

The synthesis of some deoxygenated analogues of early intermediates in the biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors.

Syntheses are described of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-d-ribo-hexopyranosyl fluoride, 6-O-acetyl-2-azido-3-O-benzyl-2,4-dideoxy-d-xylo-hexopyranosyl fluoride and 2-azido-3,4-di-O-benzyl-2,6-dideoxy-d-glucopyranosyl fluoride. These glycosyl donors were coupled with the acceptor 1d-2,3,4,5-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol and the alpha-coupled products were transformed into alpha-d-3dGlcpN-PI, alpha-d-4dGlcpN-PI and alpha-d-6dGlcpN-PI by way of the H-phosphonate route. Brief mention is made of the biological evaluation of these deoxy-sugar analogues and their N-acetylated forms as candidate substrate/inhibitors of the N-deacetylase and alpha-(1-->4)-d-mannosyltransferase activities present in trypanosomal and HeLa (human) cell-free system.

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates.

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.

Specificities of enzymes of glycosylphosphatidylinositol biosynthesis in Trypanosoma brucei and HeLa cells.

A series of synthetic analogues of d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol and the 4'-epimer, d-GalN-alpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the alpha-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the beta-linked analogue d-GlcN beta 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol alpha-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the d-myo-inositol residue, whereas the human inositol acyltransferase and/or first alpha-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place of sn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.