Monocyclic monoterpenoids carvones have been recently identified as atypical negative allosteric modulators of aryl hydrocarbon receptor (AhR). In the current work, we performed AhR antagonist activity screening of 100 natural and synthetic monoterpenoids, and their analogues. Using SAR approach, structural determinants of AhR antagonist activity were assigned, including CO presence/position, planarity, and C3/C5-alkylation. Applying pyramidal selection criteria, including absence of residual agonist activity, no cytotoxicity, strong antagonist potency, and pan-antagonism against diverse AhR agonists, we distilled four lead AhR antagonists (carvacrol, o-cresol, 3-methyl-S-carvone, EN-2). Whereas 3-methyl-S-carvone and EN-2 were non-competitive AhR pan-antagonists, carvacrol and o-cresol were ligand-selective AhR antagonists acting by unclear mechanism. We characterized in detail the effects of lead compounds at cellular functions of AhR, including AhR nuclear translocation, AhR dimerization with ARNT, and the expression of AhR-regulated genes. As a proof of concept, effects of monoterpenoids in the murine macrophages were investigated.
- MeSH
- lidé MeSH
- molekulární struktura MeSH
- monoterpeny * farmakologie chemie chemická syntéza MeSH
- myši MeSH
- receptory aromatických uhlovodíků * metabolismus antagonisté a inhibitory MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor integral to various physiological and pathological processes. Among its diverse ligands, indole-based compounds have garnered attention due to their significant biological activity and potential therapeutic applications. This study explores the activation of AhR by structurally diverse halogenated indoles. We evaluated the transcriptional activity of AhR and cell viability in the human LS174T-AhR-luc reporter cell line. Among the tested compounds, 4-FI, 7-FI, 6-BrI, 7-BrI, 6-Cl-2-ox, 5-Br-2-ox, and 6-Br-2-ox activated AhR in a concentration-dependent manner, displaying high efficacy and potency. Molecular docking analysis revealed moderate binding affinities of these compounds to the PAS-B domain of AhR, corroborated by competitive radioligand binding assays. Functional assays showed that halogenated indoles induce the formation of AhR-ARNT heterodimer and enhance the binding of the AhR to the CYP1A1 promoter. Additionally, 4-FI and 7-FI exhibited anti-inflammatory properties in Caco-2 cell models, highlighting their potential for therapeutic applications. This study underscores the significance of the type and position of halogen moiety in indole scaffold, suggesting their potential as candidates for developing therapeutics drugs to treat conditions such as inflammatory bowel disease via AhR activation.
- MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- halogenace MeSH
- indoly * chemie farmakologie MeSH
- lidé MeSH
- molekulární struktura MeSH
- receptory aromatických uhlovodíků * metabolismus chemie MeSH
- simulace molekulového dockingu * MeSH
- transkripční faktory bHLH MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 μg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.
- Publikační typ
- časopisecké články MeSH
A series of six heteroleptic copper(II) complexes with 2'-hydroxy-4-(dimethylamino)chalcone (HL) with the composition [Cu(N-N)(L)]NO3 (1-6), where N-N stands for dmbpy = 5,5'-dimethyl-2,2'-bipyridine (1), bphen = 4,7-diphenyl-1,10-phenanthroline (2), dbbpy = 4,4'-di-tert-butyl-2,2'-bipyridine (3), nphen = 5-nitro-1,10-phenanthroline (4), bpy = 2,2'-bipyridine, (5), and dpa = 2,2'-dipyridylamine (6), was prepared and thoroughly characterized. The in vitro cytotoxicity screening on eight human cancer cell lines identified complex 2, containing the bulkiest N-donor ligands (bphen) as highly cytotoxic against cancer cells, with IC50 values ranking from 1.0 to 2.3 μM, with good selectivity and low toxicity against healthy human fetal lung fibroblasts MRC-5. The cell-based assays, involving the most effective complex 2 in A2780 cancer cells, revealed its strong pro-apoptotic effects based on the effective activation of caspases 3/7, ROS overproduction, and autophagy in the A2780 cells while not impeding the cell cycle and mitochondrial membrane functions. The cellular uptake studies in A2780 and 22Rv1 cells uncovered no intracellular transport of the cationic complex 2, supporting the hypothesis that the in vitro anticancer effects of complex 2 are based on the combined extrinsic activation of apoptosis and autophagy induction.
- Publikační typ
- časopisecké články MeSH
Mycobacterial zinc metalloprotease-1 (Zmp1) is an essential enzyme for intracellular survival and pathogenicity of Mycobacterium tuberculosis. However, the exact mechanism of function of this enzyme remains unclear. This paper examines the effect of novel organic molecules on the inhibition of Zmp1. We followed our previous results and synthesised three libraries of new hydroxamates. All compounds were studied for their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis by MALDI-TOF MS. Furthermore, a macrophage infection assay was performed to evaluate intracellular antimycobacterial activity. In the whole-cell assay, no direct activity of synthesised heterocyclic hydroxamates was observed against Mycobacterium tuberculosis and Mycobacterium bovis. No acute cellular toxicity was observed against the murine RAW 264.7 macrophage cell line and human MRC-5 lung fibroblast cell line. However, thiazolidinediones 2 showed the dose-dependent inhibition of intracellular survival of Mycobacterium tuberculosis H37Ra. The inhibition was structure-dependent, with the most active derivative 2f inducing an 83.2% reduction of bacterial survival within the macrophage host cell. The promising biological activity confirmed thiazolidinediones 2 as Zmp1 inhibitors that can be used as tool compounds for further exploration of the role of Zmp1 for in vivo pathogenicity. In the long run, thiazolidinediones 2 show the potential to act as a scaffold for Zmp1 inhibitors to target intracellular Mtb as a novel tuberculosis treatment strategy.
- MeSH
- bakteriální proteiny MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- metaloproteasy metabolismus MeSH
- Mycobacterium tuberculosis * MeSH
- myši MeSH
- thiazolidindiony * farmakologie MeSH
- zinek metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This work explores the interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has a higher affinity to HSA than that of OA.
Terrestrial carnivorous plants of genera Drosera, Dionaea and Nepenthes within the order Caryophyllales employ jasmonates for the induction of digestive processes in their traps. Here, we focused on two aquatic carnivorous plant genera with different trapping mechanism from distinct families and orders: Aldrovanda (Droseraceae, Caryophyllales) with snap-traps and Utricularia (Lentibulariaceae, Lamiales) with suction traps. Using phytohormone analyses and simple biotest, we asked whether the jasmonates are involved in the activation of carnivorous response similar to that known in traps of terrestrial genera of Droseraceae (Drosera, Dionaea). The results showed that Utricularia, in contrast with Aldrovanda, does not use jasmonates for activation of carnivorous response and is the second genus in Lamiales, which has not co-opted jasmonate signalling for botanical carnivory. On the other hand, the nLC-MS/MS analyses revealed that both genera secreted digestive fluid containing cysteine protease homologous to dionain although the mode of its regulation may differ. Whereas in Utricularia the cysteine protease is present constitutively in digestive fluid, it is induced by prey and exogenous application of jasmonic acid in Aldrovanda.
Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens.
- MeSH
- bakteriální proteiny chemie genetika MeSH
- Firmicutes enzymologie genetika MeSH
- imunoglobulin A sekreční chemie MeSH
- imunoglobuliny - Fab fragmenty * chemie izolace a purifikace MeSH
- imunoglobuliny - Fc fragmenty * chemie izolace a purifikace MeSH
- lidé MeSH
- proteasy chemie genetika MeSH
- rekombinantní proteiny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 μM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).
- MeSH
- antibakteriální látky toxicita MeSH
- antimycin A analogy a deriváty toxicita MeSH
- azidy toxicita MeSH
- buněčné dýchání účinky léků MeSH
- chloramfenikol toxicita MeSH
- citrátový cyklus účinky léků MeSH
- deoxyglukosa toxicita MeSH
- fluoracetáty toxicita MeSH
- glykolýza účinky léků MeSH
- malonáty toxicita MeSH
- proteosyntéza účinky léků MeSH
- pyruváty toxicita MeSH
- reprodukovatelnost výsledků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- stav beztíže MeSH
- streptomycin toxicita MeSH
- Synechococcus chemie účinky léků izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the intracellular growth of Mycobacterium tuberculosis at a concentration of 100 and 10 μM. The thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated library.
- MeSH
- antituberkulotika chemická syntéza chemie farmakologie MeSH
- bakteriální proteiny antagonisté a inhibitory metabolismus MeSH
- kyseliny hydroxamové chemie farmakologie MeSH
- lidé MeSH
- metaloproteasy antagonisté a inhibitory metabolismus MeSH
- molekulární struktura MeSH
- Mycobacterium tuberculosis účinky léků enzymologie metabolismus MeSH
- simulace molekulového dockingu MeSH
- thiazolidindiony chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
