New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

5-Substituted Uridines with Activity against Gram-Positive Bacteria.

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. A series of uridine derivatives containing an extended substituent at the C-5 position as well as C-5 alkyloxymethyl, alkylthiomethyl, alkyltriazolylmethyl, alkylsulfinylmethyl and alkylsulfonylmethyl uridines were obtained in order to explore their antimicrobial properties and solubility. It has been shown that new ribonucleoside derivatives have an order of magnitude better solubility in water compared to their 2'-deoxy analogues and effectively inhibit the growth of a number of Gram-positive bacteria, including resistant strains of Mycobacterium smegmatis (MIC=15-200 µg/mL) and Staphylococcus aureus (MIC=25-100 µg/mL). Their activity is comparable to that of some antibiotics used in medicine.

Fungal BGCs for Production of Secondary Metabolites: Main Types, Central Roles in Strain Improvement, and Regulation According to the Piano Principle.

Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, and immunosuppressants. A single fungus species in response to various signals can produce 100 or more secondary metabolites. Such signaling is possible due to the coordinated regulation of several dozen biosynthetic gene clusters (BGCs), which are mosaically localized in different regions of fungal chromosomes. Their regulation includes several levels, from pathway-specific regulators, whose genes are localized inside BGCs, to global regulators of the cell (taking into account changes in pH, carbon consumption, etc.) and global regulators of secondary metabolism (affecting epigenetic changes driven by velvet family proteins, LaeA, etc.). In addition, various low-molecular-weight substances can have a mediating effect on such regulatory processes. This review is devoted to a critical analysis of the available data on the "turning on" and "off" of the biosynthesis of secondary metabolites in response to signals in filamentous fungi. To describe the ongoing processes, the model of "piano regulation" is proposed, whereby pressing a certain key (signal) leads to the extraction of a certain sound from the "musical instrument of the fungus cell", which is expressed in the production of a specific secondary metabolite.

Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain.

The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N1-acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.

Biotransformation of Androstenedione by Filamentous Fungi Isolated from Cultural Heritage Sites in the State Tretyakov Gallery.

The transformation of steroids by microorganisms is widely used in medical biotechnology. A huge group of filamentous fungi is one of the most promising taxa for screening new biocatalytic reactions in order to obtain pharmaceutically significant steroids. In this work, we screened 10 filamentous fungi-destructors of egg tempera for the ability to biotransform androst-4-en-3,17-dione (AD) during cultivation in a liquid nutrient medium or in a buffer solution. These taxonomically unrelated strains, belonging to the classes Eurotiomycetes, Dothideomycetes and Sordariomycetes, are dominant representatives of the microbiome from halls where works of tempera painting are stored in the State Tretyakov Gallery (STG, Moscow, Russia). Since the binder of tempera paints, egg yolk, contains about 2% cholesterol, these degrading fungi appear to be a promising group for screening for steroid converting activity. It turned out that all the studied fungi-destructors are able to transform AD. Some strains showed transformation efficiency close to the industrial strain Curvularia lunata RNCIM F-981. In total, 33 steroids formed during the transformation of AD were characterized, for 19 of them the structure was established by gas chromatography/mass spectrometry analysis. In this work, we have shown for the first time that fungi-destructors of tempera paintings can efficiently transform steroids.

Polyamines Upregulate Cephalosporin C Production and Expression of ß-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain.

The high-yielding production of pharmaceutically significant secondary metabolites in filamentous fungi is obtained by random mutagenesis; such changes may be associated with shifts in the metabolism of polyamines. We have previously shown that, in the Acremonium chrysogenum cephalosporin C high-yielding strain (HY), the content of endogenous polyamines increased by four- to five-fold. Other studies have shown that the addition of exogenous polyamines can increase the production of target secondary metabolites in highly active fungal producers, in particular, increase the biosynthesis of ß-lactams in the Penicillium chrysogenum Wis 54-1255 strain, an improved producer of penicillin G. In the current study, we demonstrate that the introduction of exogenous polyamines, such as spermidine or 1,3-diaminopropane, to A. chrysogenum wild-type (WT) and HY strains, leads to an increase in colony germination and morphological changes in a complete agar medium. The addition of 5 mM polyamines during fermentation increases the production of cephalosporin C in the A. chrysogenum HY strain by 15-20% and upregulates genes belonging to the beta-lactam biosynthetic cluster. The data obtained indicate the intersection of the metabolisms of polyamines and beta-lactams in A. chrysogenum and are important for the construction of improved producers of secondary metabolites in filamentous fungi.

Discovery of novel N4-alkylcytidines as promising antimicrobial agents.

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. In order to find new compounds that effectively inhibit the growth of pathogenic bacteria and fungi, we synthesized a set of N4-derivatives of cytidine, 2'-deoxycytidine and 5-metyl-2'-deoxycytidine bearing extended N4-alkyl and N4-phenylalkyl groups. The derivatives demonstrate activity against a number of Gram-positive bacteria, including Mycobacterium smegmatis (MIC = 24-200 µM) and Staphylococcus aureus (MIC = 50-200 µM), comparable with the activities of some antibiotics in medical use. The most promising compound appeared to be N4-dodecyl-5-metyl-2'-deoxycytidine 4h with activities of 24 and 48 µM against M. smegmatis and S. aureus, respectively, and high inhibitory activity of 0.5 mM against filamentous fungi that can, among other things, damage works of art, such as tempera painting. Noteworthy, some of other synthesized compounds are active against fungal growth with the inhibitory concentration in the range of 0.5-3 mM.

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase.

The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10-8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.