[Methodology of Screening New Antibiotics: Present Status and Prospects].

Due to extensive distribution of pathogen resistance to available pharmaceuticals and serious problems in the treatment of various infections and tumor diseases, the necessity of new antibiotics is urgent. The basic methodological approaches to chemical synthesis of antibiotics and screening of new antibiotics among natural products, mainly among microbial secondary metabolites, are considered in the review. Since the natural compounds are very much diverse, screening of such substances gives a good opportunity to discover antibiotics of various chemical structure and mechanism of action. Such an approach followed by chemical or biological transformation, is capable of providing the health care with new effective pharmaceuticals. The review is mainly concentrated on screening of natural products and methodological problems, such as: isolation of microbial producers from the habitats, cultivation of microorganisms producing appropriate substances, isolation and chemical characterization of microbial metabolites, identification of the biological activity of the metabolites. The main attention is paid to the problems of microbial secondary metabolism and design of new models for screening biologically active compounds. The last achievements in the field of antibiotics and most perspective approaches to future investigations are discussed. The main methodological approach to isolation and cultivation of the producers remains actual and needs constant improvement. The increase of the screening efficiency can be achieved by more rapid chemical identification of antibiotics and design of new screening models based on the biological activity detection.

[Irumamicin Produced by Streptomyces roseoflavus INA-1278].

The strain Streptomyces roseoflavus INA-1278 is described as a new irumamicin producer. Irumamicin 1278 is different by the antifungal activity from irumamicin produced by the world-known strain Streptomyces subflavus subsp. Irumaensis subps. nov. AM-3603.

[Microbial metabolites that inhibit sterol biosynthesis, their chemical diversity and characteristics of mode of action].

Inhibitors of sterol biosynthesis (ISB) are widespread in nature and characterized by appreciable diversity both in their chemical structure and mode of action. Many of these inhibitors express noticeable biological activity and approved themselves in development of various pharmaceuticals. In this review there is a detailed description of biologically active microbial metabolites with revealed chemical structure that have ability to inhibit sterol biosynthesis. Inhibitors of mevalonate pathway in fungous and mammalian cells, exhibiting hypolipidemic or antifungal activity, as well as inhibitors of alternative non-mevalonate (pyruvate gliceraldehyde phosphate) isoprenoid pathway, which are promising in the development of affective antimicrobial or antiparasitic drugs, are under consideration in this review. Chemical formulas of the main natural inhibitors and their semi-synthetic derivatives are represented. Mechanism of their action at cellular and biochemical level is discussed. Special attention is given to inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase (group of lovastatin) and inhibitors of acyl-CoA-cholesterol-acyl transferase (ACAT) that possess hypolipidemic activity and could be affective in the treatment of atherosclerosis. In case of inhibitors of late stages of sterol biosynthesis (after squalene formation) special attention is paid to compounds possessing evident antifungal and antitumoral activity. Explanation of mechanism of anticancer and antiviral action of microbial ISB, as well as the description of their ability to induce apoptosis is given.

[Microbial test-system for screening of inhibitors of sterol biosynthesis].

New effective economic microbial test-system for screening of microbial metabolites, that are inhibitors of sterol biosynthesis, is proposed. It is based on cultivation of fungal strain Acremonium fusidioides (former Fusidium coccineum), that produces fusidic acid (fusidin) that is antibiotic of steroid structure. Great similarity of fusidic acid biosynthesis in fungous strain and cholesterol biosynthesis in human organism, coincidence of their initial steps till squalene formation, allows use A. fusidioides as a model for estimation of microbial secondary metabolites that are potential inhibitors of sterol biosynthesis. Such inhibitors in A.fusidioides model are revealed as compounds that strongly reduce fusidin production without any visible influence on fungus growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis could be successfully applied for removal of inhibition induced by some microbial metabolites. A.fusidioides test-system can be easily mechanized because of miniaturization of microbiological procedures, cultivation in 6-, 24-, or 96-well plates and usage of automatic micropipettes and dispensers. The results of this model are well correlated with the ones obtained with human cells (Hep G2 test-system, offered earlier). A.fusidioides test-system can be applied at rather early stages of screening procedures and is quite effective for testing of crude extracts of producers' culture liquid.

[Microbial model of Halobacterium salinarum for screening inhibitors of sterol biosynthesis].

A highly effective and simple microbial test system for screening inhibitors of sterol biosynthesis (ISB) is described. The system is based on cultivation of the bacterial strain Halobacterium salinarum (former Halobacterium halobium), that possesses mevalonate pathway of sterol biosynthesis and is much similar in the biosynthesis to cholesterol formation in humans. In the H. salinarum test system the ISB were found as compounds that inhibited the test culture growth. Mevalonate which is one of the crucial intermediates of sterol biosynthesis dismissed the inhibitory effect of many microbial metabolites thus being evident of their action at the early stages of the sterol biosynthesis, including the HMG-CoA reductase stage. The H. salinarum test system was developed as a micromethod and could be easily mechanized by miniaturization of the microbiological procedures, cultivation in sterile 96-well plates and using automatic micropipettes and dispensers. The H. salinarum test system was effective in testing crude extracts of the culture broths and advantageous at early stage of screening. The use of the H. salinarum test system was shown possible for screening antitumor antibiotics.

[Microbial models in screening of inhibitors of sterol biosynthesis].

On the base of previously developed microbial models high effective scheme for screening of inhibitors of sterol biosynthesis (ISB) is proposed. It is based on cultivation of halophilic bacteria Halobacterium salinarum (former Halobacterium halobium), possessing mevalonate pathway of sterol biosynthesis, and cultivation of fungus Acremonium fusidioides (former Fusidium coccineum), that is producer of steroid antibiotic fusidin (fusidic acid), which biosynthesis has great similarity (with coincidence of its initial steps till squalene formation) to cholesterol biosynthesis in human organism. In H. salinarum model ISB are revealed as compounds that inhibit test-culture growth, whereas in A. fusidioides test-system they are revealed as compounds that strongly reduce fusidin production without any visible influence on producer's growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis remove inhibition induced by many microbial metabolites that is the evidence of their action at early stages of sterol biosynthetic pathway, including HMG-CoA reductase step. Both test-systems are developed as micromethod and could be easily mechanized due to miniaturization of microbiological procedures, cultivation in sterile 96-well plates and usage of automatic micropipettes and dispensers. Effectiveness of both test-systems, as well as their sensitiveness, laboriousness and ability to give false-positive or false-negative results in ISB screening work is compared. The proposed scheme of screening of ISB includes microbial models at early steps of screening procedures and Hep G2 test-system at the late step. The preliminary screening of microbial metabolites possessing antifungal activity at initial step is compulsory. Miniaturization and mechanization of microbial processes and purification of producers' culture broth with micro- and ultrafiltration are under consideration as well.

[Microbial model Halobacterium salinarum in screening of synthetic analogues of antibiotic turbomycin A with anticancer activity].

The microbial test-system based on cultivation of Halobacterium salinarum developed earlier for screening inhibitors of sterol biosynthesis and proposed for screening anticancer antibiotics, proved to be efficient in revealing anticancer compounds among derivatives of tris(1-alkylindol-3-yl)methylium, synthetic analogues of antibiotic turbomycin A. Most of the methane sulfonate and chloride salts of such compounds, investigated with the help of the H. salinarum test-system, showed no activity (MIC>32 mcM), while several derivatives, containing N-butyl or N-pentyl substituents were rather active against the bacterial strain. The MICs of them against H. salinarum were 8 mcM for total and 1 mcM for partial inhibition of the bacterial growth. The results of the study correlated with the results of other investigations that revealed anticancer activity of such compounds in tumor cell cultures. Therefore, the H. salinarum test-system demonstrated its availability for screening compounds with anticancer activity.

[Isolation and study of antibiotic INA-1132 (chlorothricin), produced by actinomycete strain].

Taxonomic properties of strain INA-1132 producing antibiotic INA-1132 are described. The antibiotic showed activity against grampositive bacteria and fungi. The strain was classified as belonging to the genus Streptomyces and by its taxomic characteristics is most close to S. baarnensis. The experiments with the bacterial culture Halobacterium salinarum (previously H. halobium) revealed hypolipidemic activity of the antibiotic, i. e. its ability to inhibit biosynthesis of sterols. Conditions for the production of the antibiotic, methods of its isolation and purification, as well as the results of the chemical structure elucidation are described. By its physicochemical properties the antibiotic is identical to chlorothricin. The structure of antibiotic INA-1132 was ascertained by X-ray analysis. Conformation of the molecule of chlorothricin (antibiotic 1132) was determined for the first time.

[Glycopeptide antibiotics resistance mechanism as the basis for novel derivatives development capable to overcome resistance]. / Mekhanizm rezistentnosti k glikopeptidnym antibiotikam kak osnova sozdaniia novykh proizvodnykh, sposobnykh k preodoleniiu rezistentrnosti.

The data on the genetic and biochemical bases of bacterial resistance to antibiotics of the vancomycin-ristocetin group are summarized. Special emphasis is placed on the mechanism of resistance associated with target modification and on molecular interactions occurring upon contact of glycopeptides with normal and modified targets. The prospects for developing new derivatives active against resistant bacteria are discussed. The most rational approach to the chemical transformation of glycopeptides involves the modification of the internal "binding pocket" and the peripheral regions of the molecule that participate in the stabilization of the antibiotic-target complex. Novel semisynthetic drugs of this group active against glycopeptide-resistant enterococci are described.

[Hypolipidemic action of ascofuranone in hepatoblastoma G2 cell culture]. / Gipolipidemicheskoe deistvie askofuranona v kul'ture kletok gepatoblastomy G2.

Ascofuranone, an isoprenoid antibiotic, suppressed 14C acetate incorporation into cholesterol, cholesterol ethers, triglycerides, phospholipids and free fatty acids in Hep G2 cell culture. Such a complex action of the antibiotic on lipid synthesis and metabolism was not connected with the inhibition of protein synthesis and the antibiotic toxicity.

[Increase in eremomycin production by regeneration and UV-irradiation of Amycolatopsis orientalis subsp. eremomycini protoplasts]. / Uvelichenie produktsii éremomitsina v rezul'tate regeneratsii i UF-oblucheniia protoplastov Amycolatopsis orientalis subsp. eremomycini.

Protoplast regeneration of Amycolatopsis orientalis subsp. eremomycini producing eremomycin leads to the change of cultural and morphological properties as well as synthesis of secondary metabolites. Formation of plus-variants with enchanced antibiotic production was promoted by UV-irradiation of protoplasts. These plus-variants can be successfully used for repeating protoplasting--UV-irradiation of protoplasts with further increasing of the strain productivity. Finally activity of the initial A. orientalis culture was increased 7-8 times. Proposed method is recommended for the improvement of actinomycetes strains producing antibiotics especially in the case of cultures with poor sporulation.

[The cytotoxicity of mevalonate, lovastatin and carminomycin with respect to MOLT-4 human malignant T-lymphoblasts in vitro]. / Tsitotoksichnost' mevalonata, lovastatina i karminomitsina v otnoshenii chelovecheskikh zlokachestvennykh T-limfoblastov MOLT-4 in vitro.

It was shown in vitro that high concentrations of lovastatin, a competitive inhibitor of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibited human malignant cells MOLT-4. The activity of lovastatin in doses of 50-250 microM was dose-dependent. Addition of mevalonate in a concentration of 3 mM to the growth medium completely prevented the cytotoxic effect of 100 microM of lovastatin. At the same time, exogenous mevalonate did not decrease the cytotoxicity of the anthracycline antibiotic carminomycin. Moreover, in a high concentration (7 mM) mevalonate slowly but significantly inhibited the growth of the malignant target cells and the effect was added to the cytotoxic effect of carminomycin low concentrations (0.08 to 0.175 microgram/ml). The results and the literature data suggested that combination of mevalonate, HMG-CoA reductase inhibitors and anthracyclines could be useful in tumor chemotherapy. The suggestion needs further investigation.

[Screening of microbial secondary metabolites inhibiting cholesterol biosynthesis with the use of hepatoblastoma G2]. / Otbor mikrobnykh vtorichnykh matabolitov--ingibitorov biosinteza kholesterina s pomozh'iu kul'tury kletok gepatoblastomy G2.

The culture of hepatoblastoma G2 (Hep G2) cells is proposed as an effective model for screening of microbial metabolites--inhibitors of sterol biosynthesis. This model can be applied at early stages of screening procedures and is quite effective for testing of crude extracts of producers' culture broth. The test is based on measurement inhibition of the radiolabelled precursors incorporation in cholesterol and separate fractions of lipids by microbial metabolites in Hep G2 cells. That allows not only to reveal inhibitors of cholesterol biosynthesis, but also to evaluate mechanism of action, including ability to inhibit the synthesis of cholesterol ethers. The cholesterol biosynthesis inhibition was tested at 150 microbial cultures (actinomycetes and imperfect fungi), isolated from soil. The ability to inhibit 14C-acetate incorporation into cholesterol was found in 15-20% of microbial cultures possessing antifungal activity of extracts (culture broth and mycelium).

[The hypolipidemic action of antibiotic 86/88 (enniatin B) in a hepatoblastoma G2 cell culture]. / Gipolipidemicheskoe deistvie antibiotika 86/88 (énniatina B) v kul'ture kletok gepatoblastomy G2.

A cyclodepsipeptide antibiotic 86/88 (enniatin B) with strong hypolipidemic action was isolated from the culture liquid of the fungus INA F-86/88 identified as Fusarium lateritium Nees var. stilboides (Wr.) Bilai. In the Hep G2 cell culture the antibiotic suppressed 14C-acetate incorporation into cholesterol (IC50 1.75 microM), cholesterol ethers (IC50 1 microM), triglycerides (IC50 1.3 microM) and free fatty acids (IC50 2.2 microM). The most pronounced effect of the drugs, i.e. the suppression of the cholesterol ethers synthesis is likely due not only to the ACAT inhibition but also to the inhibition of the triglyceride synthesis and the diminishing of the free fatty acids pool in the cells.

[Carminomycin induction of single-stranded DNA breaks in Micrococcus luteus cells]. / Induktsiia karminomitsinom odnonitevykh razryvov DNK kletok Micrococcus luteus.

The effect of carminomycin, an amtitumor antibiotic from the anthracycline group on DNA of M. luteus cells was studied. It was shown that carminomycin induced one-thread breaks in DNA. The antibiotic effect was proportional to its concentration and depended on the time of the cell exposure. When the incubation time with carminomycin was longer, there was observed disappearance of a significant part of the breaks. The lower was the antibiotic concentration, the rapider was the process, especially after removal of the antibiotic from the medium. Induction of the one-thread breaks by carminomycin in DNA of the cells of the mutant strain DB-7 of M. luteus was more difficult than in that of the cells of the wild type strain which was indicative of the enzymatic character of most of the breaks and of a special role of UV-endonuclease of M. luteus in this process. On the basis of carminomycin hypersensitivity of the mutant it was concluded that the above enzyme was probably involved in reparation of the DNA damages induced by carminomycin. No two-thread breaks induced by the antibiotic were detected.

[Carminomycin induction of single-stranded DNA breaks in the normal and tumorous cells of mice administered the antibiotic intravenously]. / Issledovanie induktsii karminomitsinom odnonitevykh razryvov DNK v normal'nykh i opukholevykh kletkakh myshi pri vnutrevennom vvedenii antibiotika.

The effect of carminomycin administered intravenously to albino mice on the sedimentation character of the ascitic cell DNA of mouse lymphadenosis NK/Li, as well as the cells of the lungs, kidneys, liver, heart and spleen in alkaline sucrose gradients was studied. It was found that carminomycin in a dose of 3.5 mg/kg (LD50) induced DNA single-strand breaks in the cells of the lungs, kidneys, liver and heart. At the same time the single-strand breaks were not detected in the tumor cells NK/Li and the cells of the spleen. The results are indicative of correlation between sensitivity of the cells to carminomycin and a decreased capacity of the antibiotic for induction of single-strand breaks in DNA. Probably, the cell ability for DNA repair is an important factor defining the antitumor selective effect of carminomycin. Induction of the single-strand breaks reflects the role of the excision repair in healing the damages induced by carminomycin.