Biohimiâ

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism cessation, growth rate control, virulence, bacterial persistence, and biofilm formation. The RelA/SpoT homologs superfamily proteins are responsible for (p)ppGpp alarmone synthesis, including long bifunctional RSH proteins and small alarmone synthetases. This study employs enzyme kinetics and dose-dependent inhibition methods to investigate the specific mechanism of action of DMNP involving Rel_Msm and RelZ proteins, which are (p)ppGpp synthetases in Mycolicibacterium smegmatis belonging to both types, as well as Rel_Mtb protein from Mycobacterium tuberculosis. The compound DMNP has demonstrated its capability to inhibit the activity of the Rel_Mtb protein. According to enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor targeting the Rel_Msm and RelZ proteins. Molecular docking analysis allowed to localize the DMNP binding site in proximity to the (p)ppGpp synthetase domain active site. This study advances the development of alarmone synthetase inhibitor class of compounds, which includes relacin and its derivatives, alongside the investigated compound DMNP – a synthetic analog of the marine coral metabolite erogorgiaene. Unlike the conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining the conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may potentially address prevailing challenges associated with antimicrobial resistance and bacterial persistence.

Ionic liquids (ILs) are organic salts with a low melting point, which is due to the fact that alkyl side chains chemically bonded to the ion hinder the crystallization of ILs. The low melting point of ILs has led to their widespread use as relatively harmless solvents. However, ILs have toxic properties, the mechanism of which is largely unknown, so identifying the cellular targets of ILs is of practical importance. In our work, we showed that imidazolium ILs are not able to penetrate model membranes without damaging them. We also found that inactivation of multidrug resistance (MDR) pumps in yeast cells does not increase their sensitivity to imidazolium ILs. The latter indicates that the target of the toxicity of imidazolium ILs is not in the cytoplasm. Thus, our data suggest that the disruption of the plasma membrane barrier function is the main cause of the toxicity of low concentrations of imidazolium ILs. We also showed that supplementation with imidazolium ILs restores the growth of cells with kinetically blocked glycolysis. Our data indicate that IL-induced minor disruption of the plasma membrane may, in some cases, be beneficial for the cells.

Focal adhesions (FAs) are mechanosensory structures that can convert physical stimuli into chemical signals guiding cell migration. There is a postulated correlation between FA features and cell motility parameters for individual migrating cells. However, which FA properties are essential for the movement of epithelial cells within a monolayer remains poorly elucidated. We used real-time cell visualization to describe the relationship between FA parameters and migration of immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) under inhibition or depletion of the FA proteins vinculin and FAK. To evaluate the relationship between FA morphology and cell migration, we used substrates of different elasticity in a wound healing assay. High FAK and vinculin mRNA expression, as well as largest FAs and maximal migration rate were described for cells on fibronectin, whereas cells plated on glass had minimal FA area and decelerated speed of migration into the wound. Both for normal and tumor cells, suppression of vinculin expression resulted in decreased FA size and fluorescence intensity, but had no effect on cell migration into the wound. Suppression of FAK expression or inhibition of FAK activity had no effect on FA size, but decreased FA lifetime and significantly slowed the rate of wound healing both for HaCaT and A549 cells. Our data indicates that FA lifetime, but not FA area is essential for epithelial cell migration within a monolayer. The effect of FAK kinase on the rate of cell migration within the monolayer makes FAK a promising target for antitumor therapy of lung adenocarcinoma.

Some tricyclic antidepressants (TCA), including amitriptyline (ATL), clomipramine (CLO), and desipramine (DES), are known to be effective for the management of neuropathic pain. It was previously determined that ATL, CLO and DES are capable of voltage-dependent blocking of NMDA receptors of glutamate (NMDAR), which play a key role in the pathogenesis of neuropathic pain. Despite the similar structure of ATL, CLO and DES, efficacy of their interaction with NMDAR varies significantly. In the study presented here, we applied the molecular modeling methods to investigate the mechanism of binding of ATL, CLO and DES to NMDAR and to identify the structural features of the drugs that determine their inhibitory activity against NMDAR. Molecular docking of the studied TCA into the NMDAR channel was performed. The conformational behavior of the obtained complexes in lipid bilayer was simulated by the method of molecular dynamics (MD). A single binding site (upper) for the tertiary amines ATL and CLO and two binding sites (upper and lower) for the secondary amine DES were identified inside the NMDAR channel. The upper and lower binding sites are located along the channel axis at different distances from the extracellular side of the plasmatic membrane (PM). MD simulation revealed that the position of DES in the lower site is stabilized only in the presence of sodium cation inside the NMDAR channel. DES binds more strongly to NMDAR compared to ATL and CLO due to the simultaneous interaction of two hydrogen atoms of its cationic group with the asparagine residues of the ion pore of the receptor. This feature may be responsible for stronger side effects of DES. It has been hypothesized that ATL binds to NMDAR less efficiently compared to DES and CLO due to its lower conformational mobility. The identified features of the structure- and cation-dependent mechanism of interaction between TCA and NMDAR will help in the further development of effective and safe analgesic therapy.

The study analyzed the homocysteine, cyanocobalamin (vitamin B12), folic acid (vitamin B9) and pyridoxine (vitamin B6) levels in the blood of children with confirmed demyelinating lesions of the central nervous system. The genotypes of the main polymorphisms of the folate cycle genes such as C677T and A1298C of the MTHFR gene, A2756G of the MTR gene and A66G of the MTRR gene were determined. A comparison of the studied parameters was carried out for five groups: a control group – healthy children under 18 years old, children at the onset of MS (with disease duration of no more than six months), healthy adults without neurological pathology, adult patients with MS at the onset stage of the disease and patients with long-term MS. A significant increase in homocysteine amount was revealed in children at the onset of MS compared to healthy children of the corresponding age. A high predictive value of determining homocysteine levels in children has been established. It has been demonstrated that an increased above safe values level of homocysteine is not accompanied by the presence of a vitamin deficiency state, assessed by the amount of folic acid, vitamins B6 and B12 in the blood. The lack of correlation between laboratory signs of vitamin deficiency and homocysteine levels may be due to the carriage of polymorphic variants of folate cycle genes, and an increased homocysteine level should be considered as a marker of functional disorders of folate metabolism that accompany the onset of the pathological process in MS in children. The identified patterns can be used in the development of treating strategies and preventing of the demyelination process in children with multiple sclerosis.