[A Bioinformatics Method for Identification of Human Proteases Active against Viral Envelope Glycoproteins: A Case Study on the SARS-CoV-2 Spike Protein].

Many viruses, including SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, enter host cells through a process of cell-viral membrane fusion that is activated by proteolytic enzymes. Typically, these enzymes are host cell proteases. Identifying the proteases that activate the virus is not a simple task but is important for the development of new antiviral drugs. In this study, we developed a bioinformatics method for identifying proteases that can cleave viral envelope glycoproteins. The proposed approach involves the use of predictive models for the substrate specificity of human proteases and the application of a structural analysis method for predicting the vulnerability of protein regions to proteolysis based on their 3D structures. Specificity models were constructed for 169 human proteases using information on their known substrates. A previously developed method for structural analysis of potential proteolysis sites was applied in parallel with specificity models. Validation of the proposed approach was performed on the SARS-CoV-2 spike protein, whose proteolysis sites have been well studied.

Photophysical properties and in vitro photocytotoxicity of disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX (Dimegine).

Photophysical and in vitro photocytotoxicity studies were performed for the photosensitizer Dimegine, a disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX with very low systemic toxicity. The singlet oxygen and luminescence quantum yield were ΦΔ = 0,65 ± 0,06, and Φƒ=0,11 ± 0,01 respectively, and were independent of the excitation wavelength. The photobleaching coefficients for Dimegine dissolved in phosphate buffer (pH 7.4), and DMEM medium at concentration 2 µM/l and in phosphate buffer (pH 7.0) at concentration 10 µM/l were 16·10-5, 9·10-5 and 2·10-5 respectively. In vitro cellular distribution and photocytotoxicity was studied in two human (U87 - primary glioblastoma and HT1376 - bladder cancer) and two rat cell lines (RG2 - glioma, and AY27 - bladder carcinoma). Fluorescence microscopy analysis shows primary Dimegine accumulation as small fluorescent inclusion bodies around the nuclei, suggesting an apoptotic over a necrotic cell death mechanism. The PDT efficacy was slightly higher for the rat cell lines over the human-derived cell lines, with LD50 values of 2,5 µM/l, 2.8 µM/l, 4.5 µM/l, 2.8 µM/l using 530 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively, and 1.8 µM/l, 2 µM/l, 5 µM/l, 2.4 µM/l using 625 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively. Comparison to literature data showed that Dimegine demonstrated improved phototherapeutic characteristics comparing to PpIX-mediated PDT.

[The neurotoxicity biomarkers as potential indicators of the spinal cord ischemia]. / Biomarkery neirotoksichnosti kak potentsial'nye indikatory ishemii spinnogo mozga.

The study aimed at assessing the level of glutamate receptors antibodies (Abs) in blood serum and cerebrospinal fluid (CSF) of patients with spinal cord ischemia along with traditional diagnostic approaches. MATERIAL AND METHODS: Forty patients with spinal cord ischemia (10 with spinal stroke and 30 with subacute and chronic course of the disease) were enrolled. After exclusion of some participants, 27 patients continued the study. Comparison groups included 30 patients with ischemic stroke and 30 patients with radiculopathy. The control group consisted of 15 healthy volunteers. All participants underwent a neurological examination and spinal cord magnetic resonance imaging (MRI). Abs to glutamate receptors (NR2 subunits of NMDA-receptors, AMPA/kainate receptors) were measured by ELISA. RESULTS: NR2 Abs in patients with spinal cord ischemia were significantly increased in serum (p=0.0001) and CSF (p=0.0005) compared to controls and comparison groups. The NR2 Abs reliably differentiated spinal cord ischemia compared to AMPA/kainate receptors and S100ß protein. On the other hand, increased levels of Abs to the AMPA/kainate have been detected in patients with a more severe impairment associated with extensive white matter damage. CONCLUSION: The results show the potential of the Abs to glutamate receptors assessment in the diagnosis of spinal cord ischemia and severity of the process.

[The administration of neurocytoprotectors in a rat model of experimental spinal cord ischemia]. / Primenenie neirotsitoprotektorov v usloviiakh éksperimental'noi ishemii spinnogo mozga u krys.

AIM: To study an effect of cortexin on functional recovery and morphology of the spinal cord of rats with spinal cord ischemia. MATERIAL AND METHODS: Spinal cord ischemia was achieved by ligation of the infrarenal abdominal aorta in 16 rats stratified into two equal groups: the ligation of infrarenal aorta was performed in the control group, aorta ligation was performed also in the experimental group with preliminary intraperitoneally administration of cortexin in a dose of 0.15 mg/kg 30 min before procedure. Evaluation of neurologic deficit was performed by the Tarlov's scale. Morphological evaluation was made by analyzing the histological sections of the lumbar and sacral cord using the Nissl's method of coloring. Statistical analysis was performed as well. RESULTS AND CONCLUSION: A pronounced and significant effect of cortexin, which was clinically expressed in a decrease in neurological deficit (p=0.0095), morphologically in an increase in the number of normochromic neurons (р=0.01), and a decrease in shrunken neurons (р=0.0001) and shadow cells (р=0.0003), was noted. The results suggest a potential myeloprotective effect of cortexin. The drug can be considered in the context of treatment of vascular myelopathy.

[The biomarkers of cerebral ischemia as a new method for the validation of the efficacy of cytoprotective therapy]. / Biomarkery ishemii golovnogo mozga kak novyi metod dokazatel'stva éffektivnosti neirotsitoprotektorov.

AIM: To study blood plasma concentrations of NR2-peptide in patients with ischemic stroke (IS) to assess its diagnostic value as a biomarker of cerebral ischemia and determine the dynamics of the biomarker during treatment with cortexin. MATERIAL AND METHODS: One hundred and twenty patients, aged from 18 to 70 years, including 36 with transient ischemic attack (TIA) and 84 with IS in the carotid territory (n=70) and vertebral/basilar territory with the Wallenberg-Zakharchenko syndrome (n=14), were enrolled. The National Institute of Health Stroke scale (NIHSS) was used to assess neurological status. Blood plasma concentration of NR2-peptide was measured in all patients at admission and after treatment. All laboratory results were compared with neuroimaging (MRI, CT) data. RESULTS: Concentrations of NR2-peptide detected in all patients were higher than in controls (>1.5 ng/ml), p<0.0001. The direct correlation between NR2-peptide (from 3.38 ng/ml to 15.6 ng/ml) and ischemic lesion (from few to 80 mm) was observed. A decrease in NR2-peptide concentration (from 8.5 to 5,.9 ng/ml, p<0.0001) was noted in patients treated with cortexin after 10-day treatment course. CONCLUSION: NR2-peptide blood assay is a reliable hemotest of brain ischemia. Cortexin has a sufficient therapeutic efficacy.

[EFFECT OF LOW-INTENSITY RED LASER ON GROWTH OF STAPHYLOCOCCUS AUREUSAND SENSITIZING EFFECT OF PHOTODITAZIN].

AIM: Study the effect of laser emission in the red spectrum on growth of methicillin- sensitive. and methicillin-resistant strains of Staphylococcus aureus, as well as photodynamic effect of photosensitizer photoditazin. MATERIALS AND METHODS: Effect of light of semiconduc- tor red laser (X 660 nm, 100 mW/cm2) at 30,'60, 90 and 180 J/cm2 on growth of S. aureus colonies was determined. Time of exposure 5; 10, 15 and 30 minutes. In certain series of experiments bacterial cells were sensitized in advance by a Water. solution of photoditazin at a concentration of5xl0-6 M. RESULTS: Red laser emission was established to cause a pronounced suppression of bacterial growth. This effect on standard S. aureus strain only took place dur- ing use of relatively high exposure doses (180 J/cm2). Photosensitivity of methicillin-resistant strain turned out to be significantly higher: bacteriostatic effect of red light was noted already at the dose of 60 J/cm2. Treatment of bacterial cells with photoditazin in advance signifi- cantly enhanced growth-inhibiting effect of laser light.

[Perspectives of disodium salt 2.4-di(1-metoxyethyl)-deuteroporphyrin - IX ("Dimegin") application for photodynamic therapy in non-oncologic cases].

Effects of disodium salt 2,4-di(1-metoxyethyl)-deuteroporphyrine-IX (Dimegin) and the light from Soret band (¼395-405 nm) at the viability of microbial cells and at their potential to form microbial biofilms have been compared with traditional antiseptics. Irradiation of microbial cells of S. aureus, E. coli, C. albicans and others with diode light (power density 0.05 Wt/cm2) caused a bactericidial effect similar to that obtained with standard anticeptics (chlorhexidine and dioxidine). A comparative study of the effectiveness of Dimegin and Photoditazine (a soluble salt of chlorine e6) as photosensitizers have been performed using the test system of erythrocyte hemolysis in vitro under irradiation with light from the Sore band. Results have shown insignificant difference in the photodynamic effect with similar doses of absorbed light and preparation concentration.

[Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect].

The recombinant producer of Rhodospirillum rubrum L-asparaginase (RrA) was received and purification procedure of RrA was developed. It was shown that RrA has following biochemical and catalytic characteristics: K(m) for L-asn 0.22 MM, pH optimum 9.2; temperature optimum 54 degrees C; pI = 5.1 +/- 0.3; L-gln activity seems to be low-to-negligible. K562, DU145 and MDA-MB-231 cellular lines displayed significant sensitivity towards the enzyme (IC50 = 1.80; 9.19 and 34.62 ME/ml, respectively. In comparison with L-asparaginases from E. coli II type (EcA) and Erwinia carotovora (EwA) cytotoxicity of RrA seems to be higher than EwA, but lower than EcA. 10-fold i.p. RrA administration (4000 ME/kg per day) in L5178y bearing mice showed T/C = 172%. The received results show that RrA belongs to I type cellular L-asparaginases with low L-gln activity and the high antiproliferative effect.

[Effective photosensibilizator selection for E.N.T.-organ diseases treatment, based on their accumulation in pathologically changed tissues].

Accumulation of photosensibilisators - derivatives of E6 chlorines ("Radachlorine", "Photoditazine", "Zelevsky's balsam") in the mucous membrane and selection of most effective sources of emission have been investigated in 30 patients with rhinosinusitis and 10 with tonsillitis. As a source of emission we used light emitting diode (LED) matrix device "ACT" (wavelength approximately 405 nm (Sore band)) and a laser device LAHTA-"MILON"-ML500-SP (wavelength 662 nm). Drug accumulation in the mucous membrane and changes of their concentrations after emission were evaluated by changes of fluorescence, measured with a LESA-01-BIOSPEC spectrometer. The percent of fluorescence decrease ranged from 50% to 92.7%. This suggests intensive disintegration of photosensibilisators, and consequently, high therapeutic activity of this method. Effectiveness of this method is also confirmed by clinical results.

[Long-term prognosis in patients with acute non-ST-segment elevation myocardial infarction depending on dynamics of tissue myocardial dopplerography].

AIM: To study long-term prognosis in patients with non-ST elevation acute myocardial infarction (AMI) with reference to changes in myocardial tissue dopplerography (MTD) in the course of treatment. MATERIAL AND METHODS: MTD echocardiography was conducted in 88 non-ST elevation AMI (mean age 58.0-9.8 years) and 34 healthy volunteers (mean age 58.0 +/- 9.8 years). Measurements were made of the velocity of systolic, early and late diastolic peaks at 4 levels of interventricular septum, anterior, lateral and inferior walls of the left ventricle (LV). MTD was repeated before the discharge from hospital. The patients were followed up for 10-18 months after the discharge. RESULTS: By MTD results the patients were divided into 3 subgroups: 1--an asymmetric decrease of MTD values--17(19.3%) patients who had a 20% reduction of the systolic and early diastolic peak velocity compared to healthy controls on one or two adjacent LV walls; subgroup 2--a diffuse decline of MTD values--61 (69.3%) patients. Their velocity of systolic and early diastolic peaks was subnormal on all the walls, all levels of estimation; subgroup 3--10 (11.4%) patients without MTD changes. These proportions changed in the course of treatment: the number of patients with a diffuse decrease of MTD values reduced to 31 (35.3%), the number of patients with an asymmetric MTD decrease rose to 37 (42%), and with unchanged MTD rose to 20 (22.7%) patients. The rate of development of congestive cardiac failure (CCF) and asymptomatic LV dysfunction in the long-term period was significantly higher in the subgroup with retained diffuse decrease of MTD values. CONCLUSION: The treatment of non-ST elevation AMI reduces the number of patients with a diffuse decrease of MTD values and elevates the number of patients with asymmetric decrease of MTD and unchanged MTD. Persistence of MTD diffuse changes is an unfavourable prognostic factor in relation to CCF and LV silent dysfunction.

[Inhibition of no-dependent soluble human platelet guanylate cyclase by isatin].

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 M) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.

[Study of photodynamic treatment for P-388 lympho-leukemia in mice].

Data are presented on the results of photodynamic treatment (PDT) of mice DBA2 with transplantable lympho-leukemia P-388. Different regimens of photosensitizer Dimegin and emission were used. Both intravenous PDT and in combination with local PDT should be recommended.

Analysis of close proximity quenching of phosphorescent metalloporphyrin labels in oligonucleotide structures.

Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.

YC-1-like potentiation of NO-dependent activation of soluble guanylate cyclase by derivatives of protoporphyrin IX.

The influence of protoporphyrin IX derivatives--2,4-di(1-methoxyethyl)-deuteroporphyrin IX disodium salt (dimegin) and hematoporphyrin IX (HP)--on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside was investigated. Dimegin and HP, like 1-benzyl-3-(hydroxymethyl-2-furyl)indazole (YC-1), produce synergistic effects on the activation of soluble guanylate cyclase by sodium nitroprusside. The synergistic activation of the enzyme by the combination of 10 microM sodium nitroprusside and 5 microM dimegin (or 5 microM HP) was 190 +/- 19 and 134 +/- 10%, respectively. The synergistic activation of guanylate cyclase by 3 microM YC-1 and 10 microM sodium nitroprusside was 255 +/- 19%. Dimegin and HP had no effect on the activation of guanylate cyclase by YC-1; they did not change the synergistic effect of YC-1 (3 microM) and sodium nitroprusside (10 microM) on guanylate cyclase activity. The synergistic activation of NO-stimulated guanylate cyclase activity by dimegin and HP represents a new biochemical effect of these compounds that may have important pharmacotherapeutic and physiological significance.

Photodynamic effect of novel chlorin e6 derivatives on a single nerve cell.

Chlorin e(6) and its derivatives are promising sensitizers for photodynamic therapy (PDT). In order to compare the photodynamic effects of 8 novel derivatives of chlorin e(6) and to explore some mechanisms of their effects at the cellular level, we studied PDT-induced changes in bioelectric activity of crayfish mechanoreceptor neuron that was used as a sensitive experimental model. Neurons were insensitive to red laser irradiation (632.8 nm; 0.3 W/cm(2)) or to photosensitizers alone, but changed firing rate and died under the photodynamic effect of nanomolar concentrations of sensitizers. The dynamics of neuron responses depended on photosensitizer type and concentration. The dependence of neuron lifetime on photosensitizer concentration allowed comparing efficiencies of different photosensitizers. Radachlorin was the most potent photosensitizer comparable with mTHPC. High photodynamic efficiency of some chlorin e(6) derivatives was related to weak dependence of neuron lifetime on sensitizer concentration, indicating to the initiation of 2-3 secondary processes such as free radical membrane damage by one absorbed photon. Photodynamic efficiency of sensitizers depended on amphiphilicity influencing their intracellular localization.

[Photohemolysis sensitized by deuteroporphyrin-IX derivatives: determination of binding strength of dyes to erythrocytes]. / Fotogemoliz, sensibilizirovannyi proizvodnymi deiteroporfirina-IX: opredelenie prochnosti sviazyvaniia krasitelei s éritrotsitami.

A comparative analysis of the ability of 4-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (I) and 2,4-di-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (II) to photosensitize hemolysis of human erythrocytes was performed. The photohemolytic efficiency of dye I was shown to be about 60 times higher than that of dye II. It was found that a part of each dye tightly binds to erythrocyte membranes and is not removed by washing. A method for estimating the share of the dye tightly bound to the membrane (beta) was proposed, which takes into account the shielding effect produced by the free dye and the photohemolytic efficiency of the bound dye. It was shown that the beta values for dyes I and II are 86 and 61% and correlate with the coefficients of distribution of the dyes in the octanol/water system (20.7 and 17.0, respectively).

Spectral-luminescent study of the porphyrin-diketones and their complexes.

Syntheses of octaethylporphine-diketone (OEPDK) and its platinum(II) and palladium(II) complexes (PtOEPDK, PdOEPDK) were optimized, and the dyes were isolated in a pure form in preparative quantities. They were characterized by the NMR, UV-VIS absorption and emission spectroscopy. Electronic spectra of these dyes (absorption and luminescence) were investigated in detail, and compared to corresponding porphyrins and porphyrin-monoketones. OEPDK showed a strong fluorescence at about 700 nm, while PtOEPDK and PdOEPDK showed very weak room-temperature phosphorescence in the region of 850-1100 nm and practically no fluorescence. Protonation mechanisms were studied for these dyes. Protonation at sites other than pyrrole nitrogen atoms was shown to occur, corresponding protomeric spectral forms are presented. The possibilities of the use of porphyrin-diketones as longwave fluorescent and phosphorescent probes are discussed.

Photodynamic effect of deuteroporphyrin IX and hematoporphyrin derivatives on single neuron.

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.

[Preparation and certain properties of the liposomal substance 2,4-di(1-methyl-3-hydroxybutyl)deuteroporphyrin-IX]. / Poluchenie i nekotorye svoistva liposomnogo preparata 2,4-di(1-metil-3-gidroksibutil)deiteroporfirina-IX.

A liposome preparation of a porphyrin photosensitizer for photodynamic therapy of tumors (PDT) was obtained. The in vitro efficiency of the photosensitizer was enhanced 2.5-fold through the liposome formulation. The composition and some properties of the new preparation were studied. An algorithm for a complex approach to the prediction of photosensitizer efficiencies by model experiments in vitro was developed. This approach is based on the use of two models: the determination of coefficient of distribution between n-octanol and a phosphate buffer, pH 7.4, and the determination of the cytotoxic effect on the culture of CaOv ovarian adenocarcinoma cells.

Photosensitization of skin-derived cell lines by Dimegin [2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX] in vitro.

The deuteroporphyrin-IX derivative Dimegin [2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX] was investigated with respect to cellular uptake, intracellular localization and cell survival following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry, we determined the cellular fluorescence as a marker of the uptake of Dimegin after incubation for 24 h. The intracellular localization of Dimegin was analysed using fluorescence microscopy and co-staining with fluorescent dyes specific for cell organelles. Following irradiation with an incoherent light source (580-740 nm) using a light dose of 24 J/cm2, phototoxicity was determined by means of trypan blue dye exclusion, MTT assays and growth curves. The relative Dimegin fluorescence of the different cell lines declined as follows: SCL1 > HaCaT > N1 > SCL2. Intracellular localization of Dimegin was found in the mitochondria. For all cell lines Dimegin concentrations above 15 microM yielded a significant phototoxic effect. The EC50 for SCL1 cells was 8.9 +/- 2.0 microM Dimegin. The EC50 for the cell lines increased as follows: SCL1 < HaCaT < N1 < SCL2, thus correlating with the cellular fluorescence of Dimegin. The results of the MTT assay were confirmed by trypan blue dye exclusion assay and growth curves. In conclusion, the study shows that Dimegin is an effective photosensitizer with a rapid mechanism of action in vitro, resulting in an immediate loss of plasma membrane integrity following irradiation.