Synthesis, Structure, Biological Activity, and Luminescence Properties of a "Butterfly"-Type Silver Cluster with 3-Benzyl-4-phenyl-1,2,4-triazol-5-thiol.

A new silver(I) cluster [Ag8L4(Py)(Pype)]·4Py·11H2O (I) with 3-benzyl-4-phenyl-1,2,4-triazol-5-thiol (L) was synthesized via the direct reaction of AgNO3 and L in MeOH, followed by recrystallization from a pyridine-piperidine mixture. The compound I was isolated in a monocrystal form and its crystal structure was determined via single crystal X-ray diffraction. The complex forms a "butterfly" cluster with triazol-5-thioles. The purity of the silver complex and its stability in the solution was confirmed via NMR analysis. Excitation and emission of the free ligand and its silver complex were studied at room temperature for solid samples. The in vitro biological activity of the free ligand and its complex was studied in relation to the non-pathogenic Mycolicibacterium smegmatis strain. Complexation of the free ligand with silver increases the biological activity of the former by almost twenty times. For the newly obtained silver cluster, a bactericidal effect was established.

A functional study of the global transcriptional regulator PadR from a strain Streptomyces fradiae-nitR+bld, resistant to nitrone-oligomycin.

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 «bald¼ 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.

Synthesis and antimycobacterial activity of N-(2-aminopurin-6-yl) and N-(purin-6-yl) amino acids and dipeptides.

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.

Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain.

A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F0F1 ATFase ATPase; however, 33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotoxicity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the search of intracellular molecules beyond F0F1 ATP synthase relevant to the cytotoxic properties of this perspective chemical class.

Kanamycin and Ofloxacin Activate the Intrinsic Resistance to Multiple Antibiotics in Mycobacterium smegmatis.

Drug resistance (DR) in Mycobacterium tuberculosis is the main problem in fighting tuberculosis (TB). This pathogenic bacterium has several types of DR implementation: acquired and intrinsic DR. Recent studies have shown that exposure to various antibiotics activates multiple genes, including genes responsible for intrinsic DR. To date, there is evidence of the acquisition of resistance at concentrations well below the standard MICs. In this study, we aimed to investigate the mechanism of intrinsic drug cross-resistance induction by subinhibitory concentrations of antibiotics. We showed that pretreatment of M. smegmatis with low doses of antibiotics (kanamycin and ofloxacin) induced drug resistance. This effect may be caused by a change in the expression of transcriptional regulators of the mycobacterial resistome, in particular the main transcriptional regulator whiB7.

Novel Derivatives of Quinoxaline-2-carboxylic Acid 1,4-Dioxides as Antimycobacterial Agents: Mechanistic Studies and Therapeutic Potential.

The World Health Organization (WHO) reports that tuberculosis (TB) is one of the top 10 leading causes of global mortality. The increasing incidence of multidrug-resistant TB highlights the urgent need for an intensified quest to discover innovative anti-TB medications In this study, we investigated four new derivatives from the quinoxaline-2-carboxylic acid 1,4-dioxide class. New 3-methylquinoxaline 1,4-dioxides with a variation in substituents at positions 2 and 6(7) were synthesized via nucleophilic aromatic substitution with amines and assessed against a Mycobacteria spp. Compound 4 showed high antimycobacterial activity (1.25 µg/mL against M. tuberculosis) and low toxicity in vivo in mice. Selection and whole-genomic sequencing of spontaneous drug-resistant M. smegmatis mutants revealed a high number of single-nucleotide polymorphisms, confirming the predicted mode of action of the quinoxaline-2-carboxylic acid 1,4-dioxide 4 as a DNA-damaging agent. Subsequent reverse genetics methods confirmed that mutations in the genes MSMEG_4646, MSMEG_5122, and MSMEG_1380 mediate resistance to these compounds. Overall, the derivatives of quinoxaline-2-carboxylic acid 1,4-dioxide present a promising scaffold for the development of innovative antimycobacterial drugs.

Synthesis and Characterization of Novel 2-Acyl-3-trifluoromethylquinoxaline 1,4-Dioxides as Potential Antimicrobial Agents.

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.

What are the prospects for using complexes of copper(ii) and zinc(ii) to suppress the vital activity of Mycolicibacterium smegmatis?

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 (1), [Zn2(fur)4(NH2py)2] (2, NH2py = 3-aminopyridine), [Zn(fur)2(neoc)] (3, neoc = 2,9-dimethyl-1,10-phenantroline), [Zn(OAc)2(neoc)] (4, OAc = acetat-anion), and [Cu(fur)2(neoc)(H2O)] (5) were synthesized. The structures of the compounds were established by single crystal X-ray diffraction analysis. Complexes 1 and 2 are binuclear; whereas 3-5 are mononuclear. The stabilization of supramolecular architectures in crystals for compounds 1-5 occurs due to π-π-bonding between heterocycles and hydrogen interactions that provide good solubility in aqueous solutions. The stability of the complexes upon dissolution in 5% dextrose and 0.9% NaCl was confirmed by UV-vis spectroscopic and NMR (1H) data. The study of in vitro biological activity was carried out against the non-pathogenic strain of Mycolicibacterium smegmatis that is a model for M. tuberculosis. The synergistic effect of ligands is observed for complexes 3-5 and is characterized by an increase in the biological activity values. On passage from Zn2+ to Cu2+ complexes, the biological activity increases and the maximum effect is observed for compound [Cu(fur)2(phen)]. Analysis of the transcriptomic profiles of the M. smegmatis mc 2 155 strain under the pressure of the copper complex [Cu(fur)2(phen)] made it possible to isolate 185 genes, one quarter of which are associated with the compensation of iron deficiency in the bacterial strain. Genes associated with the transport and metabolism of heavy metals, biosynthesis of fatty and amino acids, biodegradation and transport of urea were also isolated.

Bioinformatics analysis of genes of Streptomyces xinghaiensis (fradiae) ATCC 19609 with a focus on mutations conferring resistance to oligomycin A and its derivatives.

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.

Synthesis, antimicrobial and antiproliferative properties of epi-oligomycin A, the (33S)-diastereomer of oligomycin A.

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.

Sequencing and Analysis of Three Mycobacterium tuberculosis Genomes of the B0/N-90 Sublineage.

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.

Draft Genome Sequence of Streptomyces xinghaiensis (fradiae) OlgR, a Strain Resistant to Oligomycin 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.

Mutations in Efflux Pump Rv1258c (Tap) Cause Resistance to Pyrazinamide, Isoniazid, and Streptomycin in M. tuberculosis.

Although drug resistance in Mycobacterium tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be 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 in M. tuberculosis. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the M. tuberculosis Rv1258c mutants. Interestingly, the V219A and S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis. While V219A point mutation conferred low-level drug 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. The 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 resistance, 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 in M. tuberculosis and address their role in affecting treatment outcome in vivo.

Synthesis and antimycobacterial activity of imidazo[1,2-b][1,2,4,5]tetrazines.

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.

Verification of oligomycin A structure: synthesis and biological evaluation of 33-dehydrooligomycin 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.

Draft Genome Sequence of Streptomyces fradiae olg1-1, a Strain Resistant to Nitrone-Oligomycin.

We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR.

Resistance to pyrazinamide in Russian Mycobacterium tuberculosis isolates: pncA sequencing versus Bactec MGIT 960.

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.

Study on retroaldol degradation products of antibiotic oligomycin A.

Studies of reactivity of antibiotic oligomycin A in various alkaline conditions showed that the compound easily undergoes retroaldol degradation in ß-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.

Draft Genome Sequence of Streptomyces fradiae ATCC 19609, a Strain Highly Sensitive to Antibiotics.

We report here a sequence of the genome of the Streptomyces fradiae ATCC 19609 strain, initially isolated from the soil, which produces tylosin. S. fradiae is highly sensitive to different classes of antibiotics, compared to the sensitivities of other bacteria. We have identified 9 groups of genes directly or indirectly involved in the resistome formation.

A novel acyclic oligomycin A derivative formed via retro-aldol rearrangement of oligomycin A.

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.