New coumarin linked thiazole derivatives as antimycobacterial agents: Design, synthesis, enoyl acyl carrier protein reductase (InhA) inhibition and molecular modeling.

Tuberculosis is a global serious problem that imposes major health, economic and social challenges worldwide. The search for new antitubercular drugs is extremely important which could be achieved via inhibition of different druggable targets. Mycobacterium tuberculosis enoyl acyl carrier protein reductase (InhA) enzyme is essential for the survival of M. tuberculosis. In this investigation, a series of coumarin based thiazole derivatives was synthesized relying on a molecular hybridization approach and was assessed against thewild typeMtb H37Rv and its mutant strain (ΔkatG) via inhibiting InhA enzyme. Among the synthesized derivatives, compounds 2b, 3i and 3j were the most potent against wild type M. tuberculosis with MIC values ranging from 6 to 8 µg/ mL and displayed low cytotoxicity towards mouse fibroblasts at concentrations 8-13 times higher than the MIC values. The three hybrids could also inhibit the growth of ΔkatGmutant strain which is resistant to isoniazid (INH). Compounds 2b and 3j were able to inhibit the growth of mycobacteria inside human macrophages, indicating their ability to penetrate human professional phagocytes. The two derivatives significantly suppress mycobacterial biofilm formation by 10-15 %. The promising target compounds were also assessed for their inhibitory effect against InhA and showed potent effectiveness with IC50 values of 0.737 and 1.494 µM, respectively. Molecular docking studies revealed that the tested compounds occupied the active site of InhA in contact with the NAD+ molecule. The 4-phenylcoumarin aromatic system showed binding interactions within the hydrophobic pocket of the active site. Furthermore, H-bond formation and π -π stacking interactions were also recorded for the promising derivatives.

Identification of new anti-mycobacterial agents based on quinoline-isatin hybrids targeting enoyl acyl carrier protein reductase (InhA).

Tuberculosis (TB) is a global issue that poses a significant economic burden as a result of the ongoing emergence of drug-resistant strains. The urgent requirement for the development of novel antitubercular drugs can be addressed by targeting specific enzymes. One such enzyme, Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein (enoyl-ACP) reductase (InhA), plays a crucial role in the survival of the MTB bacterium. In this research study, a series of hybrid compounds combining quinolone and isatin were synthesized and assessed for their effectiveness against MTB, as well as their ability to inhibit the activity of the InhA enzyme in this bacterium. Among the compounds tested, 7a and 5g exhibited the most potent inhibitory activity against MTB, with minimum inhibitory concentration (MIC) values of 55 and 62.5 µg/mL, respectively. These compounds were further evaluated for their inhibitory effects on InhA and demonstrated significant activity compared to the reference drug Isoniazid (INH), with IC50 values of 0.35 ± 0.01 and 1.56 ± 0.06 µM, respectively. Molecular docking studies investigated the interactions between compounds 7a and 5g and the target enzyme, revealing hydrophobic contacts with important amino acid residues in the active site. To further confirm the stability of the complexes formed by 5g and 7a with the target enzyme, molecular dynamic simulations were employed, which demonstrated that both compounds 7a and 5g undergo minor structural changes and remain nearly stable throughout the simulated process, as assessed through RMSD, RMSF, and Rg values.

Heterologous overexpression and preliminary antimicrobial activity test of salmocin M, a novel colicin M-like bacteriocin against Salmonella sp.

Currently, it is extremely important to identify and describe new alternative compounds with potential antimicrobial properties. Since various natural biological systems are capable of producing active compounds with such properties, many of them have been the subject of intensive study. The aim of this work was to heterologously overexpress, purify and preliminarily investigate the antimicrobial activity of a novel bacteriocin found in Salmonella species. Overexpressed protein shows an amino acid structure homologous to the well-known colicin M and was never expressed previously in the E. coli platform. Purified salmocin M showed an inhibition spectrum against Salmonella and E. coli strains. To determine its potential as an antimicrobial agent for use in medicine or the food industry, preliminary antimicrobial tests against pathogenic bacteria were carried out. Our research demonstrates that bacteriocin can be produced efficiently in bacterial expression systems, which are one of the cheapest and the most popular platforms for recombinant protein production. Moreover, preliminary results of microbiological tests showed its activity against most of the bacterial strains in a dose-dependent manner.

1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis.

1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

Evaluation of the Mycobactericidal Effect of Thio-functionalized Carbohydrate Derivatives.

Sugars with heteroatoms other than oxygen have attained considerable importance in glycobiology and in drug design since they are often more stable in blood plasma due to their resistance to enzymes, such as glycosidases, phosphorylases and glycosyltransferases. The replacement of oxygen atoms in sugars with sulfur forms thio-sugars, which are potentially useful for the treatment of diabetes and some bacterial and viral infections. Here, we evaluated the antibacterial activity of thio-functionalized carbohydrate derivatives. A set of 21 compounds was screened against acid-fast Mycobacterium tuberculosis (Mtb), gram-negative Escherichia coli and gram-positive Staphylococcus aureus. The tested carbohydrate derivatives were most effective against tubercle bacilli, with as many as five compounds (thioglycoside 6, thiosemicarbazone 16A, thiosemicarbazone 20, aminothiadiazole 23, and thiazoline 26) inhibiting its growth with MIC50 ≤ 50 µM/CFU. Only two compounds (aminothiadiazole 23 and thiazoline 26) were able to inhibit the growth of E. coli at concentrations below 1 mM, and one of them, aminothiadiazole 23, inhibited the growth of S. aureus at a concentration ≤1 mM. The five compounds affecting the growth of mycobacteria were either thiodisaccharides (6, 16A, and 20) or thioglycosides (23 and 26). All of these compounds (6, 16A, 20, 23, and 26) were able to inhibit the growth of Mtb deposited within human macrophages. However, three of the five selected compounds (6, 23, and 26) exhibited relatively high cytotoxicity in mouse fibroblasts at micromolar concentrations. The selected thio-sugars are very promising compounds, thus making them candidates for further modifications that would decrease their cytotoxicity against eukaryotic cells without affecting their antimycobacterial potential.

Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli.

The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use ß-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5'-triphosphate. This fact leads to the conclusion that NAD⁺-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD⁺ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD⁺-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

Principles of a New Protocol for Prediction of Azole Resistance in Candida albicans Infections on the Basis of ERG11 Polymorphisms.

In recent years, Candida albicans infections treatment has become a growing problem because, among others, pathogenic strains are capable to develop resistance to the administered drugs. The elaboration of rapid and accurate method of resistance assessment is an important goal of many studies. They aim to avoid inappropriate dosage or drug choice, which may be life threatening in case of severe candidiasis. Here we propose a new protocol to predict C. albicans infections. The resistance prediction is based on high-resolution melt (HRM) analysis of ERG11 gene, especially, at the particularly unstable regions. Two statistically significant nucleotide polymorphisms were detected among twenty-seven strains isolated from saliva, one of which was silent mutation (Glu266Asp, Leu480Leu). We propose also HRM analysis as a convenient, simple and inexpensive method of preliminary selection of C. albicans DNA samples that vary in ERG11 nucleotide sequence within presumed region. Taken together, our study provides firm basis for the development of fast, simple and reliable methodology for diagnosis of C. albicans infections.

Genetic variation of the ABC transporter gene ABCC1 (Multidrug resistance protein 1-MRP1) in the Polish population.

BACKGROUND: Multidrug resistance-associated protein 1 (MRP1), encoded by the ABCC1 gene, is an ATP-binding cassette transporter mediating efflux of organic anions and xenobiotics; its overexpression leads to multidrug resistance. In this study, 30 exons (from 31 in total) of the ABCC1 gene as well as and their flanking intron sequences were screened for genetic variation, using the High Resolution Melting (HRM) method, for 190 healthy volunteers representing the Polish population. Polymorphism screening is an indispensable step in personalized patient therapy. An additional targeted SNP verification study for ten variants was performed to verify sensitivity of the scanning method. RESULTS: During scanning, 46 polymorphisms, including seven novel ones, were found: one in 3' UTR, 21 in exons (11 of them non-synonymous) and 24 in introns, including one deletion variant. These results revealed some ethnic differences in frequency of several polymorphisms when compared to literature data for other populations. Based on linkage disequilibrium analysis, 4 haplotype blocks were determined for 9 detected polymorphisms and 12 haplotypes were defined. To capture the common haplotypes, haplotype-tagging single nucleotide polymorphisms were identified. CONCLUSIONS: Targeted genotyping results correlated well with scanning results; thus, HRM is a suitable method to study genetic variation in this model. HRM is an efficient and sensitive method for scanning and genotyping polymorphic variants. Ethnic differences were found for frequency of some variants in the Polish population compared to others. Thus, this study may be useful for pharmacogenetics of drugs affected by MRP1-mediated efflux.

Evaluation of DNA primase DnaG as a potential target for antibiotics.

Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

The MM2QM tool for combining docking, molecular dynamics, molecular mechanics, and quantum mechanics.

The use of the MM2QM tool in a combined docking + molecular dynamics (MD) + molecular mechanics (MM) + quantum mechanical (QM) binding affinity prediction study is presented, and the tool itself is discussed. The system of interest is Mycobacterium tuberculosis (MTB) pantothenate synthetase in complexes with three highly similar sulfonamide inhibitors, for which crystal structures are available. Starting from the structure of MTB pantothenate synthetase in the "open" conformation and following the combined docking + MD + MM + QM procedure, we were able to capture the closing of the enzyme binding pocket and to reproduce the position of the ligands with an average root mean square deviation of 1.6 Å. Protein-ligand interaction energies were reproduced with an average error lower than 10%. The discussion on the MD part and a protein flexibility importance is carried out. The presented approach may be useful especially for finding analog inhibitors or improving drug candidates.

Depletion of tRNA CCA-adding enzyme in Mycobacterium tuberculosis leads to polyadenylation of transcripts and precursor tRNAs.

In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase (rv3907c, PcnA) as a functional CCA-adding enzyme (CCAMtb) essential for viability of M. tuberculosis. The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCAMtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.

ATP-Dependent Ligases and AEP Primases Affect the Profile and Frequency of Mutations in Mycobacteria under Oxidative Stress.

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.

Comprehensive Analysis of ABCG2 Genetic Variation in the Polish Population and Its Inter-Population Comparison.

ATP-binding cassette sub-family G member 2 (ABCG2), also known as breast cancer resistance protein (BCRP), is one of the key efflux ATP-binding cassette (ABC) transporters of xenobiotics, their metabolites and endogenous compounds such as urate. Some of its genetic variants have been found to influence protein functioning, resulting in serious clinical implications concerning chemotherapy response, as well as gout or blood group phenotype Jr(a-). Previous reports have suggested that the frequencies of certain crucial polymorphisms, such as c.34G>A (p.Val12Met) and c.421C>A (p.Gln141Lys) differ significantly between the Polish population and other Caucasian populations. Thus, to clarify this issue, the present study performs a complete analysis of the genetic variation of ABCG2 coding sequence in the Polish population. The genetic variation in 14 out of 15 coding exons of the ABCG2 gene, as well as their flanking intron sequences, were examined among 190 healthy representatives of the Polish population using scanning with High Resolution Melting (HRM). HRM scanning revealed 17 polymorphisms: eight in the exons (including five missense variants and one point-nonsense mutation) and nine in the intron sequences (eight single nucleotide polymorphisms (SNPs) and one deletion variant). These included variants correlating with the presence of gout and phenotype Jr(a-). Linkage disequilibrium, haplotype blocks and haplotype analyses were also performed. The frequencies of the most common polymorphisms in the Polish population did not differ significantly to those observed for other Caucasian populations, but demonstrated divergence from non-Caucasian populations. We hope that our findings may be helpful for other researchers and clinicians, evaluating the pharmacogenetic role of ABCG2.

Molecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C.

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.

Novel Isoniazid-Carborane Hybrids Active in Vitro Against Mycobacterium tuberculosis.

Tuberculosis (TB) is a severe infectious disease with high mortality and morbidity. The emergence of drug-resistant TB has increased the challenge to eliminate this disease. Isoniazid (INH) remains the key and effective component in the therapeutic regimen recommended by World Health Organization (WHO). A series of isoniazid-carborane derivatives containing 1,2-dicarba-closo-dodecaborane, 1,7-dicarba-closo-dodecaborane, 1,12-dicarba-closo-dodecaborane, or 7,8-dicarba-nido-undecaborate anion were synthesized for the first time. The compounds were tested in vitro against the Mycobacterium tuberculosis (Mtb) H37Rv strain and its mutant (DkatG) defective in the synthesis of catalase-peroxidase (KatG). N'-((7,8-dicarba-nido-undecaboranyl)methylidene)isonicotinohydrazide (16) showed the highest activity against the wild-type Mtb strain. All hybrids could inhibit the growth of the ΔkatG mutant in lower concentrations than INH. N'-([(1,12-dicarba-closo-dodecaboran-1yl)ethyl)isonicotinohydrazide (25) exhibited more than 60-fold increase in activity against Mtb DkatG as compared to INH. This compound was also found to be noncytotoxic up to a concentration four times higher than the minimum inhibitory concentration 99% (MIC99) value.

PPE51 Is Involved in the Uptake of Disaccharides by Mycobacterium tuberculosis.

We have recently found that selected thio-disaccharides possess bactericidal effects against Mycobacterium tuberculosis but not against Escherichia coli or Staphylococcus aureus. Here, we selected spontaneous mutants displaying resistance against the investigated thio-glycoside. According to next-generation sequencing, four of six analyzed mutants which were resistant to high concentrations of the tested chemical carried nonsynonymous mutations in the gene encoding the PPE51 protein. The complementation of these mutants with an intact ppe51 gene returned their sensitivity to the wild-type level. The uptake of tritiated thio-glycoside was significantly more abundant in wild-type Mycobacterium tuberculosis compared to the strain carrying the mutated ppe51 gene. The ppe51 mutations or CRISPR-Cas9-mediated downregulation of PPE51 expression affected the growth of mutant strains on minimal media supplemented with disaccharides (maltose or lactose) but not with glycerol or glucose as the sole carbon and energy source. Taking the above into account, we postulate that PPE51 participates in the uptake of disaccharides by tubercle bacilli.

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation.

The physiological role of the non-homologous end-joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (DeltarecA, Deltaku, DeltaligD, Deltaku/ligD, DeltarecA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ-mutant strains (Deltaku, DeltaligD, Deltaku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis. However, there was little difference in IR sensitivity between NHEJ-mutant and wild-type strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A DeltarecA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the DeltarecA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively.

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment.

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.

Mycobacterial DNA Replication as a Target for Antituberculosis Drug Discovery.

BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. FACTORS: Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. DISCUSSION AND CONCLUSION: This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and elongation of the DNA during the replication process. They are pivotal processes required for successful multiplication of the bacterial cells and hence they are extensively investigated for the development of antituberculosis drugs. Finally, we summarize the most potent inhibitors of DNA synthesis and provide an up to date report on their status in the clinical trials.

Functional Analysis of the rs774872314, rs116171003, rs200231898 and rs201107751 Polymorphisms in the Human RORγT Gene Promoter Region.

RAR-related orphan receptor gamma RORγT, a tissue-specific isoform of the RORC gene, plays a critical role in the development of naive CD4+ cells into fully differentiated Th17 lymphocytes. Th17 lymphocytes are part of the host defense against numerous pathogens and are also involved in the pathogenesis of inflammatory diseases, including autoimmune disorders. In this study, we functionally examined four naturally occurring polymorphisms located within one of the previously identified GC-boxes in the promoter region of the gene. The single nucleotide polymorphisms (SNPs) rs774872314, rs116171003 and rs201107751 negatively influenced the activity of the RORγT promoter in a gene reporter system and eliminated or reduced Sp1 and Sp2 transcription factor binding, as evidenced by the electrophoretic mobility shift assay (EMSA) technique. Furthermore, we investigated the frequency of these SNPs in the Polish population and observed the presence of rs116171003 at a frequency of 3.42%. Thus, our results suggest that polymorphisms within the RORγT promoter occurring at significant rates in populations affect promoter activity. This might have phenotypic effects in immune systems, which is potentially significant for implicating pathogenetic mechanisms under certain pathological conditions, such as autoimmune diseases and/or primary immunodeficiencies (e.g., immunoglobulin E (IgE) syndrome).