Investigation of 5’-norcarbocyclic nucleoside analogues as antiprotozoal and antibacterial agents

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein

PLGA Carriers for Controlled Release of Levofloxacin in Anti-Tuberculosis Therapy

Levofloxacin (LFX) is a highly effective anti-tuberculosis drug with a pronounced bactericidal activity against Mycobacterium tuberculosis (Mtb). In this work, an “organic solvent-free” approach has been used for the development of polylactic-co-glycolic acid (PLGA) microparticles and scaffolds containing LFX at a therapeutically significant concentration, providing for its sustained release. To achieve the target, both nonpolar supercritical carbon dioxide and polar supercritical trifluoromethane have been used. By changing the composition, surface morphology, size, and internal structure of the polymer carriers, one can control the kinetics of the LFX release into phosphate buffered saline solutions and physiological media, providing for its acceptable burst and desirable concentration in the prolonged phase. The biocompatibility and bactericidal efficacy of PLGA/LFX carriers assessed both in vitro (against Mtb phagocytosed by macrophages) and in vivo (against inbred BALB/c mice aerogenically infected with Mtb) demonstrated their anti-tuberculosis activity comparable with that of the standard daily intragastric levofloxacin administration. These results make it possible to consider the developed compositions as a promising candidate for anti-tuberculosis control release formulations providing for the further evaluation of their activity against Mtb and their metabolism in vivo over long periods of tuberculosis infection

The type II TA systems of mycobacteria were investigated. Schematic diagram of the toxin-antitoxin system.

<p>(A) TA systems are annotated according to the GenBank database, excluding VapBC50 (rv3750c-rv3749c), VapBC49 (rv3180c-rv3181c), HigBA3 (rv3182-rv3183), HigBA2 (rv2022c-rv2021c), MazEF10 (rv0298-rv0299) and VapBC45 (rv2018-rv2019) systems; these systems are annotated according to Sala et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]. The system RelBE3 (rv3358-rv3357, GenBank database, NCBI) is called the YefM/YoeB system by Sala. All of the TA systems depicted here are type II (systems marked with an asterisk are novel TA systems that are not classified to any family, but for which functional activity has been shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.ref032" target="_blank">32</a>]). The 13 genes, our proposed set for genotyping, are highlighted in bold. (B) Type II TA systems are encoded by two genes, a toxin and an antitoxin, that form one operon with a promoter located upstream of the first antitoxin gene. PIN domain is the functional part of the toxin gene, the four conserved acidic residues marked at the picture: the three well-conserved acidic residues, at positions 4[D], 40[E] and 93[D], and with fourth acidic residue is less well conserved at position 112[D].</p

Novel 5′-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents

A series of novel 5&#8242;-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 &#956;g/mL (mc2155) and a MIC99 of 6.7&#8315;67 &#956;g/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28&#8315;61 &#956;g/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50&#8315;60 &#956;g/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC99 20&#8315;50 &#956;g/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20&#8315;50 &#956;g/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls

<i>Mycobacterium tuberculosis</i> Type II Toxin-Antitoxin Systems: Genetic Polymorphisms and Functional Properties and the Possibility of Their Use for Genotyping

<div><p>Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of <i>Mycobacterium tuberculosis</i> in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of <i>M</i>. <i>tuberculosis</i> was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of <i>M</i>. <i>tuberculosis</i> strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of <i>M</i>. <i>tuberculosis</i>. The possibility of using our set of genes for genotyping using PCR is also demonstrated.</p></div

Scheme of typing of <i>M</i>. <i>tuberculosis</i> strains using 13 genes of type II TA systems.

<p>The algorithm for determining the genotype is presented. The scheme shows that, after the first iteration to determine the genotype, the number of genes for the analysis is decreased twofold. Each gene in the brackets is given its position that is replaced, and the appropriate nucleotide is indicated. All replacements are calculated relative to the reference strain H37Rv.</p

Detection of the Ural genotype by qPCR.

<p>Fluorescence in the FAM channel (blue): (1)13_2978, (2) 13–3114, (3) 13–3086, (4) 13_3158, (5) 13_4178, (6) 13_3539, (7) 13_2566, (8) 13_3632, (9) 13_3599, (10) 13_3896, (11) 13_3582, (12) 13_4189, (13) 13_3535, (15) 13_3147; Fluorescence in the HEX channel (green): (14) 13_3147, (16) 13_2978. Fluorescence of the channel FAM (blue) indicates the accumulation of the PCR product containing cytosine (C); the fluorescence of the channel HEX (green) indicates the accumulation of the PCR product containing thymine (T, the variable nucleotide) and indicates the SNP in the <i>vapC10</i> gene (C394→T394) characteristic of the Ural genotype. Line 14 (13_3147) and 16 (13_2978) belong to the Ural genotype. For isolate 13_2978 fluorescence is detected on the two channels (FAM and HEX), this can indicate the presence of impurities (coinfection). qPCR fluorescence in RFU (relative fluorescence units) vs. PCR cycles. Intensity of fluorescence depending on the number of qPCR cycles for strains belonging to the Euro-American lineage.</p

Phylogenetic relationship between different genotypes of the <i>M</i>. <i>tuberculosis</i>.

<p>(A) Phylogenetic tree constructed on the basis of polymorphisms (SNP) in all of the considered genes of type II TA systems. An unrooted phylogenetic tree for the 173 strains from this study was constructed based on the presence/absence of SNPs in the nucleotide sequences of 71 TA systems (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.s003" target="_blank">S3 Table</a>); (B) Phylogenetic tree constructed on the basis of SNP in a minimum set of genes of type II TA systems. An unrooted phylogenetic tree for 173 strains constructed based on SNPs in the nucleotide sequences of 13 genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#pone.0143682.t002" target="_blank">Table 2</a>). In both of cases strains included in the one cluster belong to the same genotype (various genotypes highlighted by color). The trees was constructed by the neighbor-joining approach. The TA systems sequences were retrieved from different databases (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143682#sec002" target="_blank">Materials and Methods</a>). Sequences were multiply aligned by using ClustalW ver. 2.1 software. The trees was calculated using MEGA ver. 6. Bootstrap support > 60% is indicated for the trees.</p