Oleandomycin and its natural and synthetic analogs. II. Structure and properties of oleandomycin B, a new compound of the oleandomycin group

The revealed regularities of mass spectroscopic disintegration of oleandomycin and its derivatives made it possible to determine analytic criteria for identification of compounds related by their structure to oleandomycin. Analysis of the extracts from oleandomycin fermentation broth filtrates on the basis of the selected group of diagnostic ions showed that along with the main antibiotic there formed during the biosynthesis oleandomycin B, a structurally close minor component. The structure of the substance was assigned and its physico-chemical and biological properties were studied.     

Drug resistance in Staphylococcus aureus. Induction of macrolide resistance by erythromycin, oleandomycin and their derivatives.

Antibacterial and inducer activities concerning inducible macrolide resistance in Staphylococcus aureus were investigated using 32 erythromycin, oleandomycin and other macrolide antibiotic derivatives and analogues. The macrolides were classified into five groups from very high to none according to their inducer activity.     

Kinetics of thermal inactivation of oleandomycin in aqueous and native alkaline solutions

The process of oleandomycin inactivation in aqueous alkaline solutions with their heating was studied by using the microbiological method of the antibiotic content assay. The initial specific rate of inactivation of crystalline oleandomycin in buffer solutions and oleandomycin in the fermentation broth filtrate was evaluated. It was shown that the inactivation was retarded by the reaction products and the components of the fermentation broth filtrate. The production rate of oleandomycin anhydro derivatives amounting to 3-40 per cent of the total mass of the inactivation product was estimated by UV spectrophotometry.     

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI , oleN2 and oleR ). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus . The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI , oleR and oleD genes were expressed in Streptomyces lividans . OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.     

Effect of hydroacridine derivatives on the sensitivity of polyresistant strains of Staphylococcus aureus and Escherichia coli to antibiotics]

Sensitivity of 4 clinical strains of Staph. aureus and E. coli to 13 hydroacridine derivatives and their combinations with antibiotics, such as benzylpenicillin, ampicillin, semi-synthetic penicillins, streptomycin, chloramphenicol, tetracycline, chlortetracycline, monomycin, oleandomycin and erythromycin was studied. The highest bacteriostatic effect was observed on the use of perhydroactidine derivatives with benzylpenicillin or ampicillin with respect to polyresistant penicillinase-producing strains of Staph. aureus, resistance of which to these antibiotics was decreased 250--1000 times. Under the effect of the above compounds the staphylococcal resistance to chloramphenicol, tetracycline, chlortetracycline, oleandomycine and erythromycin decreased 2--66 times. The combinations of hydroacridine with the antibiotics, except 10-amino-trans-syn-trans-perhydroacridine had no effect on the resistance of the E. coli strains. The results of the combined effect of the above substances were associated with their chemical nature, the bacterial type and possibly the character of the strain resistance.     

Specific drug binding to rat liver cytochrome P-450 isozymes induced by pregnenolone-16 alpha-carbonitrile and macrolide antibiotics. Implications for drug interactions

Clinical interactions of macrolides with various drugs lead to elimination impairment, increase of plasma concentration and overdose-like effects, resulting from modifications of their metabolism. Previous studies have shown that treatment of rats by the macrolide antibiotics of the oleandomycin and erythromycin series lead to the induction of an hepatic cytochrome P-450 which is implicated into their own metabolism. We have characterized PCN or macrolides induced cytochromes P-450 by their specific ability to interact with macrolide derivatives and, using the cytochrome P-450 spectral binding assays, we have shown that some compounds, implicated in drug interaction with macrolides, interact preferentially with the same cytochromes. This strongly suggests that specific blockage of cytochrome P-450 IIIA1 family by macrolides, is responsible for these drug interactions and that these interactions can be predicted easily by simple in vitro tests such as those described herein.     

Drug Resistance in Staphylococcus aureus

Antibacterial and inducer activities concerning inducible macrolide resistance in Staphylococcus aureus were investigated using 32 erythromycin, oleandomycin and other macrolide antibiotic derivatives and analogues. The macrolides were classified into five groups from very high to none according to their inducer activity.     

Generation of hybrid elloramycin analogs by combinatorial biosynthesis using genes from anthracycline-type and macrolide biosynthetic pathways

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.     

Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism, Streptomyces antibioticus.

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.     

Analysis of thiamine and its phosphate esters by high-performance liquid chromatography.

High-performance liquid chromatography was used to separate thiamine and its phosphate esters after conversion to corresponding highly fluorescent thiochrome derivatives by alkaline oxidation. These compounds were absorbed on LiChrosorb-NH₂, eluted with acetonitrile-90 mm potassium phosphate buffer (pH 8.4), and determined spectrofluorometrically. A complete, rapid, and quantitative separation of thiochrome and its phosphate derivatives was made and the minimum amount detected was 1 pmol for each of these compounds.     

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.     

Biliary excretion of foreign compounds. Benzene and its derivatives in the rat

1. The extent of the excretion in the bile of the rat of benzene and 21 of its simple derivatives was studied. 2. Some 16 compounds of molecular weight less than 200, and including neutral molecules (benzene and toluene), aromatic acids, aromatic amines and phenols, were injected in solution intraperitoneally into biliary-cannulated rats. Metabolites in the bile were identified and estimated. The extent of biliary excretion of these compounds was low, i.e. 0–10% of the dose in 24hr., and most appeared in the bile mainly as conjugates. 3. The biliary excretion of six conjugates of molecular weight less than 300, including three glycine conjugates, one sulphate conjugate, one glucuronic acid conjugate and two acetyl derivatives, was low (less than 3% of the dose). 4. It is concluded that simple benzene derivatives of molecular weight less than about 300 are poorly excreted in rat bile.     

Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics.

The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.     

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.     

Sensitivity of various races of the tularemia microbe to erythromycin, oleandomycin and polymyxin]

Sensitivity of 66 strains of the tularemia microbe to erythromycin, oleandomycin and polymyxin was studied. It was shown that all the strains were resistant to polymyxin. The strains of the non-artic and Central Asiatic races and 64 per cent of the strains of the holarctic race were resistant to erythromycin and oleandomycin.     

The interrelation between the formation of oleandomycin and the resistance to it in different strains of Streptomyces antibioticus]

Strains, producers of oleandomycin, with different level of antibiotic-formation have been studied for their resistance to their own antibiotic. The obtained highly active strain possesses double resistance to oleandomycin and 50% higher activity. Identity of oleandomycin phosphate substances synthesized by initial and produced highly active strains is shown by the HELC method.     

Effect of alpha-tocopherol and its derivatives on ATPase activity and oxidative phosphorylation in rat liver mitochondria

The effects of alpha-tocopherol and its derivatives on the energy metabolism in mitochondria were studied. It was shown that alpha-tocopherol derivatives with short hydrocarbon chain produce uncoupling effects, while alpha-tocopherol itself as well as its long-chain derivatives (tocopheryl acetate and tocopheryl stearate) exert no effect. All the derivatives studied produced an inhibitory effect on DNP-stimulated mitochondrial ATPase. As far as these compounds did not influence the activity of mitochondrial F1 factor, it was concluded that the effects of the compounds were due to membrane modification.     

Biosynthesis of oleandomycin by cultures of Streptomyces antibioticus obtained after regeneration and fusion of protoplasts

The ability of Streptomyces antibioticus strains to synthesize oleandomycin is studied under the effect of regeneration and fusion of protoplasts. The production of strains-regenerants with an increased (by 30-50%) synthesis of oleandomycin is possible. Regenerants of mutants resistant to the proper antibiotic retain a high level of the oleandomycin synthesis more stably. Variations in the antibiotic-production ability are considered in regenerant populations of various generations.     

Inhibition of leucocyte elastase by heparin and its derivatives.

Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. Ki values ranged from 40 nM to 100 microM and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di- and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established.     

Regio- and stereoselective enzymatic esterification of glycerol and its derivatives.

A methodology for regio- and stereoselective preparation of acyl glycerol derivatives is presented. It offers easy access to specific 1,2-, 1,3-diglycerides and triglycerides as well as alkyl glycerol esters, phospholipids and glycolipids. These compounds are prepared by esterification of the corresponding glycerol derivatives such as 2-monoglycerides, alkyl glycerols, glyceryl glycosides, glyceryl phosphate esters, or unsubstituted glycerol. The regio- and stereoselectivity in the esterification is achieved by using fatty acid anhydrides and an enzymatic catalyst, 1,3-specific lipase. NMR methods for determining the regio- and stereoselectivity of esterification are discussed.