Search

New complexes of zinc(ii) and copper(ii) with 2-furoic acid (Hfur), acetic acids and N-donor ligands with the compositions [Zn2(fur)4]n, [Zn2(fur)4(NH2py)2], [Zn(fur)2(neoc)], [Zn(OAc)2(neoc)], and [Cu(fur)2(neoc)(H2O)] were synthesized.

The reaction of barium(II) hydroxide with 2-furoic acid (pyromucic acid, Hfur) gave 1D coordination polymer {[Ba3(fur)6(Hfur)2(H2O)8]}n·4nH2O (I). According to X-ray diffraction data, I contains two symmetrically independent barium cations, which have different environments: Ba(1) coordinates six fur– anions, while Ba(2) is a fragment of heteroleptic coordination (fur–, Hfur, and H2O) (CIF file CCDC no. 2063380). The supramolecular polymeric architecture of I is determined by bridging water molecules and numerous hydrogen bonds between coordinated and solvation water molecules to form a 1D motif. The arrangement of Ba2+ ions in the trinuclear moiety {Ba3(fur)6(Hfur)2(H2O)8} of the polymer is nearly linear. The thermal behavior of I, investigated by simultaneous thermal analysis (argon atmosphere), showed a stepwise character of thermal decomposition caused by gradual destruction of the hydrogen-bonded framework and the subsequent partial destruction of the organic part. Analysis of the biological activity of I against the non-pathogenic Mycolicibacterium smegmatis strain showed low sensitivity of the bacterial wall to the complex.

The reaction of copper(ii) acetate with 2-furancarboxylate (Hfur, pyromucate) anions and the N-donor ligands 4-phenylpyridine (phpy) and 3-aminopyridine (NH2py) in acetonitrile afforded the mononuclear complexes of the composition [Cu(fur)2(phpy)2(H2O)] · phpy (1) and [Cu(fur)2(NH2py)2] (2), respectively. The structures of the complexes were established by X-ray diffraction. The simultaneous thermal analysis of the thermal behavior of complex 1 showed that this complex is thermally stable up to 125 °C. The in vitro biological activity of complexes 1 and 2 was evaluated against the non-pathogenic mycobacterial Mycolicibacterium smegmatis strain.

The mononuclear complexes [M(Fur)2(Bpy)(H2O)] (M = Cu (I), Zn (II)) were prepared by the reactions of copper(II) and zinc(II) acetates with 2-furoic acid (HFur) anion and 2,2'-bipyridine (Bpy) in acetonitrile. A similar reaction with ZnCl2 used instead of Zn(OAc)2 · 2H2O gave the complex [Zn(Fur)2(Bpy)] (III). The cobalt(II) complex [Co(Fur)2(Bpy)] (IV) was obtained by the reaction of cobalt trimethylacetate with HFur and Bpy. The structure of mononuclear complexes was determined by X-ray diffraction (CIF files CCDC nos. 1993168 (I), 1993169 (II), 1993170 (III), and 1993171 (IV)). Compounds I, III, and IV were studied for the biological activity in vitro against the nonpathogenic mycobacterial strain Mycolicibacterium smegmatis. It was found that I and III have high biological activity and are promising for further studies for antituberculosis activity.

The reaction of copper(II) trimethylacetate, [Cu(Piv)2]n (Piv = C(Me)3COO–), with caffeine (L) (Cu : L = 1 : 1) in anhydrous acetonitrile yielded the binuclear complex [Cu2(Piv)4(L)2] ⋅ 2CH3CN (I). The structure of the complex in the crystal was established by X-ray diffraction (CIF file CCDC no. 2 006 753), and the electronic structure was studied by ESR. The effect of caffeine coordination to the biogenic complexing agent was established: the in vitro biological activity of the obtained complex towards the non-pathogenic strain Mycolicibacterium smegmatis was eight times higher than that of free caffeine.

Objectives The aim of this study was to obtain Streptomyces xinghaiensis (fradiae) ATCC 19609 mutants resistant to oligomycin A and its derivatives and to identify the underlying mechanism of resistance. This study was based on the premise that S. xinghaiensis ATCC 19609 contains several oligomycin A biological targets, explaining why the strain remains supersensitive to oligomycin A despite all efforts to obtain resistant mutants using standard genetic methods. Methods The method to obtain oligomycin A-resistant mutants was performed in two steps: first, mutants slightly resistant to an oligomycin A derivative with an attenuated effect were obtained; and second, oligomycin A-resistant mutants were obtained from those mutants obtained earlier. The genomes of the mutants were then sequenced and a bioinformatics analysis of the detected mutations was conducted. Results Mutants with seven mutations were required to obtain oligomycin A-resistant mutant strains of S. xinghaiensis characterised by a level of resistance comparable with that of the model organism Streptomyces lividans. Five of these mutations caused amino acid substitutions in the well-known oligomycin A biological target, namely the F0F1-ATP synthase A subunit, and the others caused amino acid substitutions in unexplored biological targets, including RecB-like recombinase, type IV helicase, DNA ligase and single-domain response regulator. Conclusion A new oligomycin resistance mechanism involving a pathway that repairs double-strand breaks in DNA known as non-homologous end joining (NHEJ) was discovered.

The strain Streptomyces xinghaiensis (fradiae) ATCC19609 is sensitive to antibiotics of various chemical classes and is hypersensitive to the macrolide antibiotic oligomycin A. The spontaneous mutant S. fradiae-nitR+bld, resistant to nitron-oligomycin, carries a mutation in the gene of the PadR transcriptional regulator. A comparative proteomic analysis of the inverted membrane vesicles of the mutant strain and wild-type S. xinghaiensis (fradiae) ATCC19609 was performed. Using mass spectrometric analysis, quantitative changes in the protein fractions of two ABC transporters, leucyl aminopeptidase, alkaline phosphatase, and alanine dehydrogenase were detected in vesicles of the mutant strain compared with wild-type vesicles. As a result of transcriptional analysis, an increase in the expression levels of the genes of ABC transporters, alkaline phosphatase, and leucyl aminopeptidase and a decrease in the expression level of the alanine dehydrogenase gene in the mutant strain S. fradiae-nitR+bld compared with the wild-type strain were recorded. This suggested that the padR gene, in which the mutation was detected, may be involved in the regulation of antibiotic resistance and differentiation.

Mono- and binuclear complexes [Cu(Fur)2(Phen)] (I), [Cu2(Fur)4(Py)2] (II), [Cu(Fur)2-(Py)2(H2O)] (III), and [Zn2(Fur)4L2] (L = Py (IV), Phpy (V)) are synthesized using the reactions of copper(II) and zinc(II) acetates with anions of pyromucic acid (2-furancarboxylic acid (HFur)) and N‑donor ligands (pyridine (Py), 4-phenylpyridine (Phpy), and 1,10-phenanthroline (Phen)) in acetonitrile. The copper complexes with pyridine are successively formed from the same reaction mixture: at first binuclear complex II is formed and then mononuclear complex III is formed. All compounds are isolated as single crystals, and their structures are determined by X-ray structure analysis (CIF files CCDC nos. 1974386 (I), 1974388 (II), 1974389 (III), 1974387 (IV), and 1974385 (V)). The thermal behavior of complexes II and III is studied by simultaneous thermal analysis. The biological activity in vitro of all complexes is determined toward the nonpathogenic mycobacterial strain Mycolicibacterium smegmatis. Complexes I and V exhibit a high biological activity and are promising for further studies of antitubercular activity.

The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.

New complexes of zinc(ii) and copper(ii) with 2-furoic acid (Hfur), acetic acids and N-donor ligands with the compositions [Zn

Tuberculosis (TB) has recently become the leading killer among infectious diseases. Multidrug and extensively drug-resistant Mycobacterium tuberculosis strains urge the need to develop anti-TB drugs with a novel mechanism of action. We describe synthesis of 22 novel imidazo[1,2-b][1,2,4,5]tetrazine derivatives with different substituents at C(3) and C(6) positions, and their antimycobacterial activity in vitro. 8 compounds show activity as potential serine/threonine protein kinase (STPK) inhibitors in M. smegmatis aphVIII+ test-system, which is characteristic for this class. 3 compounds out of 5 most active STPK inhibitors have a prominent minimal inhibitory concentration on M. tuberculosis H37Rv of 1 μg/ml. We were able to obtain M. smegmatis mc2 155 mutants resistant to 4 compounds and show that they do not have cross resistance with other drugs, but have a common mechanism of resistance among these 4 imidazo[1,2-b][1,2,4,5]tetrazines. Compound 3h seems the most promising, combining a predicted STPK inhibitor activity, the lowest MIC on M. tuberculosis and a low frequency of drug resistant mutants’ emergence.

We describe the synthesis of epi-oligomycin A, a (33S)-diastereomer of the antibiotic oligomycin A. The structure of (33S)-oligomycin A was determined by elemental analysis, spectroscopic studies, including 1D and 2D NMR spectroscopy, and mass spectrometry. Isomerization of C33 hydroxyl group led to minor changes in the potency against Aspergillus niger, Candida spp., and filamentous fungi whereas the activity against Streptomyces fradiae decreased by approximately 20-fold compared to oligomycin A. We observed that 33-epi-oligomycin A had the same activity on the human leukemia cell line K562 as oligomycin A but was more potent for the multidrug resistant subline K562/4. Non-malignant cells were less sensitive to both oligomycin isomers. Finally, our results pointed at the dependence of the cytotoxicity of oligomycins on oxygen supply.

We describe Streptomyces fradiae mechanisms of sensitivity to nitrone-oligomycin A, a derivative of oligomycin A. We obtained S. fradiae-nitR+ bld, a nitrone-oligomycin A resistant mutant with a «bald» phenotype. Comparative genomic analysis of the wild-type S. fradiae ATCC19609 and S. fradiae-nitR+ bld revealed a mutation in padR - a gene encoding a multifunction transcription regulator, which resulted in the amino acid replacement in a highly conserved DNA-binding domain. Bioinformatics genome analysis of S. fradiae ATCC19609 discovered a PadR binding site 13 bp upstream the start codon of the marR transcription factor gene. Induction of S. fradiaenitR+ bld and w.t. strains with nitrone-oligomycin A lead to a significant increase in expression level of the marR gene in the w.t. strain, but no change observed in mutant strain. We identified differences between DNA-protein interactions of the mutant and native PadR proteins with its putative binding site in S. fradiae ATCC19609. This allowed us to suggest that the padR gene, that harbored a single nucleotide mutation in the S. fradiaenitR+ bld strain, might be involved in the mechanism of resistance to nitrone-oligomycin A. We assume the participation of the transcriptional factorpadR in the formation of the bald phenotype.

Studies of the antimycobacterial activity of 2-amino-6-chloropurine and its N-acyl derivatives against laboratory (Mycobacterium tuberculosis H37Rv, M. avium, M. terrae) and clinical Mycobacterium tuberculosis strains (MDR-TB) identified compounds with high tuberculostatic activity (MIC from 0.35 – 1.5 μg/ml). The cytotoxic activities of the compounds with antimycobacterial activity against human embryo fibroblasts were studied in the MTT test, which showed that the study compounds were essentially nontoxic (IC50 > 50 μM). Further chemical modification may yield compounds with potential for creating drugs for the treatment of tuberculosis. With the aim of identifying the possible mechanism of the tuberculostatic activity, the ability of the study compounds to inhibit mycobacterial serine-threonine protein kinases (STPK) was assessed. The tuberculostatic activity of 2-amino-6-chloropurine derivatives was not linked with their inhibition of STPK.

The M. tuberculosis resistome includes a number of genes involved in intrinsic drug resistance. One of the major members of this system is the whiB7 gene encoding a transcription factor, which regulates expression of a number of genes—its regulon. In this study, we analyze the impact on intrinsic drug resistance levels of the whiB7 and tap genes and their prevalent mutants. The mutations found within whiB7 and tap genes in the genomes of strains belonging to Beijing and EAI-Manila lineages allowed us to investigate the new role of these genes in the intrinsic drug resistance of mycobacteria to β-lactams, fluoroquinolones, and macrolides and to assume their significance in the development of M. tuberculosis genotypes. Here we also established that the mutation in the tap gene–insC581, previously described, as a marker of the Beijing lineage, cannot be the marker due to its absence in a number of Beijing strains.

Data of draft genome sequencing of Streptomyces fradiae-АТСС19609-Olg4R strain, resistant to (33S)-33-deoxy-33-thiocyanatooligomycin A, are presented. Comparative analysis of the genomes of S. fradiae wild-type strain ATCC19609 and S. fradiae strain ATCC19609-Olg4R showed the single nucleotide substitution that led to A(600)T change in the conservative P-loop NTPase region of class IV helicase gene, which, probably, promoted the resistance of S. fradiae strain ATCC19609-Olg4R to (33S)-33-deoxy-33-thiocyanatooligomycin A.

Although, the structure of oligomycin A (1) was confirmed by spectroscopic and chemical evaluations, some crystallographic data cast doubt on the originally adopted structure of the side 2-hydroxypropyl moiety of this antibiotic. It was suggested that the side chain of the oligomycin is enol-related (2-hydroxy-1-propenyl). To clarify this matter we synthesized and evaluated 33-dehydrooligomycin A (2) prepared by the Kornblum oxidation of 33-O-mesyloligomycin A (3) by dimethyl sulfoxide. NMR data for 33-dehydrooligomycin (2) and results of quantum chemical calculations have shown that this derivative exists in the keto rather than in the enol tautomer 2a. The in vitro antimicrobial activity of 2 was approximately two times weaker in comparison with oligomycin A against Streptomyces fradiae ATCC-19609 and reference Candida spp. strains and similar activity against certain filamentous fungi. The docking binding estimate of 2 with FOF1ATP synthase showed a slight decrease in binding affinity for 2 when compared with oligomycin A; that correlated with its activity against S. fradiae ATCC 19609 that is supersensitive to oligomycin A. The in vitro antiproliferative activities of 2 are also discussed.

In the present work, we analyze the mutant strains of Streptomyces fradiae ATCC 19609 resistant to (33S)-azido-33-deoxyoligomycin A, obtained at a frequency of 10–8 to 2.5 × 10–9. Analysis of the obtained mutant strains allows them to be divided into two groups based on the level of resistance to oligomycin A. One mutant out of 15 obtained is resistant to oligomycin A at MIC of 0.1 nm/mL, and 14 mutant strains are resistant at MIC of 0.01 nm/mL of oligomycin A (MIC for the initial strain: 0.001 nm/mL). Using PCR analysis and subsequent sequencing by the Sanger method, three candidate genes the products of which are related to resistance to oligomycin A are analyzed: helicase IV, the C subunit and the A subunit of FoF1-ATP synthase. It is found that, in the genomes of all 15 mutants, there is a single nucleotide substitution (SNP) in the helicase IV gene, while in one strain most resistant to oligomycin A there is an additional SNP in the gene of the A subunit of FoF1-ATP synthase. This mutant strain will be used to obtain the mutants of resistance to oligomycin A with the aim of further sequencing of genomes and detection of biotarget of oligomycin A.

We report the draft genome sequences of three Mycobacterium tuberculosis isolates belonging to the B0/N-90 sublineage, EKB34, EKB53, and EKB79. The B0/N-90 sublineage belongs to the prevalent (in Russia) and highly virulent Beijing-B0/W148 sublineage. Isolates EKB34 and EKB79 were obtained from people with immune deficiency.

We describe the synthesis of epi-oligomycin A, a (33

New complexes of nickel(II) with 2-furoic acid (2-Hfur) and different N-donor ligands of the composition [Ni(fur)2(phen)(H2O)2]·H2O (1) (phen = 1,10-phenantroline), [Ni(fur)2(2,2′-bpy)] (2, 2,2′-bpy = 2,2′-bipyridine), [Ni(fur)2(4,4′-bpy)(CH3OH)2]n (3, 4,4′-bpy = 4,4′-bipyridine), [Ni(fur)2(pz)4]·2MeCN (4) (pz = pyrazole) have been synthesized. The structure of the compounds was established by single crystal X-ray diffraction analysis. Complexes 1, 2, and 4 are mononuclear, complex 3 is a 1D coordination polymer. The DC magnetic susceptibility data measured for 2 and 3 are indicative of antiferromagnetic exchange interactions between the metal centers, which proved to be negligibly weak in 2 and more pronounced in 3. A quantum-chemical analysis of intermolecular (for 2) and intra- and interchain (for 3) magnetic exchange pathways confirms these findings, revealing the presence of magnetic chains formed by a network of very weak intermolecular antiferromagnetic exchange interactions in the crystal of 2 and a strengthening of antiferromagnetic exchange coupling in the polymeric chains of 3. Thermal behavior of 1 and 2 was studied by simultaneous thermal analysis (STA). According to PXRD data, the final thermolysis product of complex 2 is metal Ni nanocrystals (d = 14.7(3) nm). Analysis of the results of biological activity of 1–4 in vitro against Mycolicibacterium smegmatis showed a low sensitivity of the mycobacterial wall to Ni2+ cations.

The paper provides the annotation and data on sequencing the antibiotic resistance genes in Streptomyces fradiae strain ATCC19609, highly sensitive to different antibiotics. Genome analysis revealed four groups of genes that determined the resistome of the tested strain. These included classical antibiotic resistance genes (nine aminoglycoside phosphotransferase genes, two beta-lactamase genes, and the genes of puromycin N-acetyltransferase, phosphinothricin N-acetyltransferase, and aminoglycoside acetyltransferase); the genes of ATP-dependent ABC transporters, involved in the efflux of antibiotics from the cell (MacB-2, BcrA, two-subunit MDR1); the genes of positive and negative regulation of transcription (whiB and padR families); and the genes of post-translational modification (serine-threonine protein kinases). A comparative characteristic of aminoglycoside phosphotransferase genes in S. fradiae ATCC19609, S. lividans TK24, and S. albus J1074, the causative agent of actinomycosis, is provided. The possibility of using the S. fradiae strain ATCC19609 as the test system for selection of the macrolide antibiotic oligomycin A derivatives with different levels of activity is demonstrated. Analysis of more than 20 semisynthetic oligomycin A derivatives made it possible to divide them into three groups according to the level of activity: inactive (>1 nmol/disk), 10 substances; with medium activity level (0.05–1 nmol/disk), 12 substances; and more active (0.01–0.05 nmol/disk), 2 substances. Important for the activity of semisynthetic derivatives is the change in the position of the 33rd carbon atom in the oligomycin A molecule.

We report a draft genome sequence of Streptomyces xinghaiensis ( fradiae ) OlgR, which is resistant to oligomycin A. This mutant strain is derived from S. xinghaiensis OlgR2.100, which is resistant to (33 S )-azido-33-deoxyoligomycin A.

We report a draft genome sequence of Streptomyces xinghaiensis (fradiae) OlgR, which is resistant to oligomycin A. This mutant strain is derived from S. xinghaiensis OlgR2.100, which is resistant to (33S)-azido-33-deoxyoligomycin A., We report a draft genome sequence of Streptomyces xinghaiensis (fradiae) OlgR, which is resistant to oligomycin A. This mutant strain is derived from S. xinghaiensis OlgR2.100, which is resistant to (33S)-azido-33-deoxyoligomycin A. We have identified single nucleotide polymorphisms (SNPs) in 7 genes, which may lead to oligomycin A resistance.

Resistance to pyrazinamide (PZA) may impact clinical outcome of anti-tuberculosis chemotherapy. PZA susceptibility testing using MGIT 960 is not reliable and little information is available on the prevalence of PZA resistance in Russia. A collection of 64 clinical isolates of Mycobacterium tuberculosis, including 35 multidrug resistant and extensively drug-resistant (MDR/XDR), was analyzed for PZA resistance using MGIT 960, Wayne test, and sequencing of PZA resistance genes pncA, rpsA and panD. In addition, we analyzed 519 MDR-TB strains for susceptibility to PZA by MGIT 960. Sequencing of pncA revealed 17 of 25 (68%) MDR strains and all 10 XDR strains harboring pncA mutations. A correlation of φ = 0.81 between MGIT 960 and pncA sequencing was observed. Mutations in rpsA and panD not associated with PZA resistance as defined by MGIT 960 were identified. We found 1 PZA-resistant strain without mutations in known PZA resistance genes. Almost 73% of MDR-TB strains isolated in Moscow, Russia, were PZA-resistant by MGIT 960 testing of 519 MDR-TB clinical isolates. Further studies are needed to determine the role of rpsA and panD mutations in possible low-level PZA resistance and to identify the molecular basis of new PZA resistance in the isolate without known PZA resistance mutations.

New usnic acid (UA) derivatives were investigated in vitro to elucidate their potential inhibitory activities on the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis. Seven pairs of enantiomers of thiazole UA derivatives were tested using the M. smegmatis strain mc2 155 test system, and the “structure–activity” relationship was established. The most active compounds were (+)-3 and (−)-3, and their kinase inhibitory activities were investigated. The results obtained using the Streptomyces lividans APHVIII+ and M. smegmatis APHVIII+ test systems indicated the significant protein kinase activity of these compounds and revealed the species specificity of the actions of the dextro- and levorotatory isomers. Both isomers, (+)-3 and (−)-3, possess similar inhibitory activity against M. tuberculosis H37Rv. The action of the isomers on eukaryotic cells was also investigated, and the results demonstrate that the dextrorotatory isomer (+)-3 leads to the lysis of intact macrophages to a degree higher than that obtained spontaneously and significantly higher than that obtained with the levorotatory isomer.

Although drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

The problem of Mycobacterium tuberculosis virulence, together with drug resistance, is becoming key for the design of drugs with a new mechanism of action and the production of modern concepts and tuberculosis treatment schemes. The review describes gene complexes and their products, including mycolic acids and global regulatory systems at the level of transcriptional, translational, and post-translational modification, etc. The criteria for selection of virulence/pathogenicity factors that might be used for comparative genomic analysis of strains differing in the degree of virulence were recommended. The experimental approaches and test systems for an adequate estimation of the virulence degree of different strains of M. tuberculosis were analyzed.

Studies of reactivity of antibiotic oligomycin A in various alkaline conditions showed that the compound easily undergoes retroaldol degradation in β-hydroxy ketone fragments positioned in the C7-C13 moiety of the antibiotic molecule. Depending on reaction conditions, the retroaldol fragmentation of the 8,9 or 12,13 bonds or formation of a product through double retroaldol degradation, when the fragment C9-C12 was detached, took place followed by further transformations of the intermediate aldehydes formed. The structures of the obtained non-cyclic derivatives of oligomycin A were supported by NMR and MS methods. NMR parameters demonstrate the striking similarity of the geometry (conformation) of the fragment C20-C34 in the non-cyclic products of retroaldol degradation and the starting antibiotic 1. The compounds obtained had lower cytototoxic properties than oligomycin A for human leukemia cells K-562 and colon cancer cells HCT-116 and lower activity against growth inhibition of model object Streptomyces fradiae. It cannot be excluded that the products of retroaldol degradation participate in the biological effects of antibiotic oligomycin A.

Synthetic routes to novel N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates with amino acids and glycine-containing dipeptides were developed. In vitro testing of 42 new and known compounds made it possible to reveal a series of N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates exhibiting significant antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant M. tuberculosis strain isolated from tuberculosis patients in the Ural region (Russia). N-(2-Aminopurin-6-yl)- and N-(purin-6-yl)-glycyl-(S)-glutamic acids were the most active compounds.

The antibiotic oligomycin A in the presence of K(2)CO(3) and n-Bu(4)NHSO(4) in chloroform in phase-transfer conditions afforded a novel derivative through the initial retro-aldol fragmentation of the 8,9 bond, followed by further transformation of the intermediate aldehyde. NMR, MS and quantum chemical calculations showed that the novel compound is the acyclic oligomycin A derivative, in which the 8,9 carbon bond is disrupted and two polyfunctional branches are connected with spiroketal moiety in positions C-23 and C-25. The tri-O-acetyl derivative of the novel derivative was prepared. The acyclic oligomycin A derivative retained the ability to induce apoptosis in tumor cells at low micromolar concentrations, whereas its antimicrobial potencies decreased substantially. The derivative virtually lost the inhibitory activity against F(0)F(1) ATP synthase-containing proteoliposomes, strongly suggesting the existence of the target(s) beyond F(0)F(1) ATP synthase that is important for the antitumor potency of oligomycin A.

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on the E. coli protein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence of Streptomyces coelicolor Pk25 protein kinase. Mutant and wild-type aphVIII were cloned into E. coli with the catalytic domain of pk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants of E. coli containing both aphVIII and pk25 were more resistant to kanamycin than those carrying only aphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of PknB of Mycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F0F1 ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F0F1 complex is Mg2+-dependent and moderately stimulated by high concentrations of Ca2+ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F0F1 ATPase inhibitors that react with the membrane-bound F0 complex. The assay of biochemical properties of the F0F1 ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F0F1 complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F0F1 ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.