Klára Konečná

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β‐lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.

Sulfadiazine Salicylaldehyde-Based Schiff Bases: Synthesis, Antimicrobial Activity and Cytotoxicity

The resistance among microbes has brought an urgent need for new drugs. Thus, we synthesized a series of Schiff bases derived from the sulfa drug sulfadiazine and various salicylaldehydes. The resulting 4-[(2-hydroxybenzylidene)amino]-N-(pyrimidin-2-yl)benzene-sulfonamides were characterized and evaluated against Gram-positive and Gram-negative bacteria, yeasts, moulds, Mycobacterium tuberculosis, nontuberculous mycobacteria (M. kansasii, M. avium) and their cytotoxicity was determined. Among bacteria, the genus Staphylococcus, including methicillin-resistant S. aureus, showed the highest susceptibility, with minimum inhibitory concentration values from 7.81 µM. The growth of Candida sp. and Trichophyton interdigitale was inhibited at concentrations starting from 1.95 µM. 4-[(2,5-Dihydroxybenzylidene)amino]-N-(pyrimidin-2-yl)-benzenesulfonamide was identified as the most selective Schiff base for these strains with no apparent cytotoxicity and a selectivity index higher than 16. With respect to M. tuberculosis and M. kansasii that were inhibited within the range of 8 to 250 µM, unsubstituted 4-[(2-hydroxy-benzylidene)amino]-N-(pyrimidin-2-yl)benzenesulfonamide meets the selectivity requirement. In general, dihalogenation of the salicylic moiety improved the antibacterial and antifungal activity but also increased the cytotoxicity, especially with an increasing atomic mass. Some derivatives offer more advantageous properties than the parent sulfadiazine, thus constituting promising hits for further antimicrobial drug development.

Synthesis and biological evaluation of N-alkyl-3-(alkylamino)-pyrazine-2-carboxamides

A series of N-alkyl-3-(alkylamino)pyrazine-2-carboxamides and their N-alkyl-3-chloropyrazine-2-carboxamide precursors were prepared. All compounds were characterized by analytical methods and tested for antimicrobial and antiviral activity. The antimycobacterial MIC values against Mycobacterium tuberculosis H37Rv of the most effective compounds, 3-(hexylamino)-, 3-(heptylamino)- and 3-(octylamino)-N-methyl-pyrazine-2-carboxamides 14‒16, was 25 μg/mL. The compounds inhibited photosystem 2 photosynthetic electron transport (PET) in spinach chloroplasts. This activity was strongly connected with the lipophilicity of the compounds. For effective PET inhibition longer alkyl chains in the 3-(alkylamino) substituent in the N-alkyl-3-(alkylamino)pyrazine-2-carboxamide molecule were more favourable than two shorter alkyl chains.

Design, Synthesis, Antimycobacterial Evaluation, and In Silico Studies of 3-(Phenylcarbamoyl)-pyrazine-2-carboxylic Acids

Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56 μg·mL−1 (5 μM). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13 μg·mL−1. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-d-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding.

Synthesis of Novel Pyrazinamide Derivatives Based on 3-Chloropyrazine-2-carboxamide and Their Antimicrobial Evaluation

Aminodehalogenation of 3-chloropyrazine-2-carboxamide with variously substituted benzylamines yielded a series of fifteen 3-benzylaminopyrazine-2-carboxamides. Four compounds possessed in vitro whole cell activity against Mycobacterium tuberculosis H37Rv that was at least equivalent to that of the standard pyrazinamide. MIC values ranged from 6 to 42 µM. The best MIC (6 µM) was displayed by 3-[(4-methylbenzyl)amino]pyrazine-2-carboxamide (8) that also showed low cytotoxicity in the HepG2 cell line (IC50 ≥ 250 µM). Only moderate activity against Enterococcus faecalis and Staphylococcus aureus was observed. No activity was detected against any of tested fungal strains. Molecular docking with mycobacterial enoyl-ACP reductase (InhA) was performed to investigate the possible target of the prepared compounds. Active compounds shared common binding interactions of known InhA inhibitors. Antimycobacterial activity of the title compounds was compared to the previously published benzylamino-substituted pyrazines with differing substitution on the pyrazine core (carbonitrile moiety). The title series possessed comparable activity and lower cytotoxicity than molecules containing a carbonitrile group on the pyrazine ring.

Design, Synthesis and Evaluation of N-pyrazinylbenzamides as Potential Antimycobacterial Agents

Three series of N-(pyrazin-2-yl)benzamides were designed as retro-amide analogues of previously published N-phenylpyrazine-2-carboxamides with in vitro antimycobacterial activity. The synthesized retro-amides were evaluated for in vitro growth inhibiting activity against Mycobacterium tuberculosis H37Rv (Mtb), three non-tuberculous mycobacterial strains (M. avium, M. kansasii, M. smegmatis) and selected bacterial and fungal strains of clinical importance. Regarding activity against Mtb, most N-pyrazinylbenzamides (retro-amides) possessed lower or no activity compared to the corresponding N-phenylpyrazine-2-carboxamides with the same substitution pattern. However, the active retro-amides tended to have lower HepG2 cytotoxicity and better selectivity. Derivatives with 5-chloro substitution on the pyrazine ring were generally more active compared to their 6-cloro positional isomers or non-chlorinated analogues. The best antimycobacterial activity against Mtb was found in N-(5-chloropyrazin-2-yl)benzamides with short alkyl (2h: R2 = Me; 2i: R2 = Et) in position 4 of the benzene ring (MIC = 6.25 and 3.13 µg/mL, respectively, with SI > 10). N-(5-Chloropyrazin-2-yl)benzamides with hydroxy substitution (2b: R2 = 2-OH; 2d: R2 = 4-OH) on the benzene ring or their acetylated synthetic precursors possessed the broadest spectrum of activity, being active in all three groups of mycobacterial, bacterial and fungal strains. The substantial differences in in silico calculated properties (hydrogen-bond pattern analysis, molecular electrostatic potential, HOMO and LUMO) can justify the differences in biological activities between N-pyrazinylbenzamides and N-phenylpyrazine-2-carboxamides.

3-Substituted N-Benzylpyrazine-2-carboxamide Derivatives: Synthesis, Antimycobacterial and Antibacterial Evaluation

A series of substituted N-benzyl-3-chloropyrazine-2-carboxamides were prepared as positional isomers of 5-chloro and 6-chloro derivatives, prepared previously. During the aminolysis of the acyl chloride, the simultaneous substitution of chlorine with benzylamino moiety gave rise to N-benzyl-3-(benzylamino)pyrazine-2-carboxamides as side products, in some cases. Although not initially planned, the reaction conditions were modified to populate this double substituted series. The final compounds were tested against four mycobacterial strains. N-(2-methylbenzyl)-3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a) and N-(3,4-dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a) proved to be the most effective against Mycobacterium tuberculosis H37Rv, with MIC = 12.5 μg·mL−1. Compounds were screened for antibacterial activity. The most active compound was 3-chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (5) against Staphylococcus aureus with MIC = 7.81 μM, and Staphylococcus epidermidis with MIC = 15.62 μM. HepG2 in vitro cytotoxicity was evaluated for the most active compounds; however, no significant toxicity was detected. Compound 9a was docked to several conformations of the enoyl-ACP-reductase of Mycobacterium tuberculosis. In some cases, it was capable of H-bond interactions, typical for most of the known inhibitors.