Preclinical efficacy investigation of human neutrophil elastase inhibitor sivelestat in animal model of psoriasis.

Background: Psoriasis is a chronic immune-mediated inflammatory skin disease manifested by an increased rate of keratinocyte division. Currently, it has been established that the cytokines of the IL-36 family play a significant role in the initiation and regulation of the inflammatory process in psoriasis. The IL-36 cytokine found in skin is inactive and its activation requires proteolytic processing that may occur via the involvement of neutrophil serine proteases such as human neutrophil elastase (HNE). The localization of these enzymes in the upper layers of the epidermis suggests that topical application of HNE inhibitors could be efficacious in the treatment of psoriasis. Sivelestat is an HNE inhibitor developed for systemic use towards the treatment of acute respiratory failure. Aim: The present study focussed on the investigation of the effects of sivelestat formulated for topical use, in the imiquimod-induced model of psoriasis in mice. Methods: The psoriasis-like state was induced by application of imiquimod (Aldara®) 5% cream to mouse shaven skin. A group of 40 inbred mice of the BALB/c strain randomized into 4 groups of 10 was used in the experiment: Group 1 - no therapy (control), Group 2 - ointment (Vaseline) containing 1% sivelestat, Group 3 - cream (lanoline + olive oil + water in equal proportions) containing 1% sivelestat, Group 4 - 1% betamethasone dipropionate. Dermatological assessment of skin lesions was performed by means of the PASI method (mPASI), as well as histological and immunohistochemical evaluation. Results: Based on the evaluation of efficacy manifestations, it was established that the total mPASI index value decreased by 50% during therapy with sivelestat cream and by 36% during therapy with sivelestat ointment. Histological study revealed that the epidermal thickness in groups that underwent therapy was 2.4-3.6 times lower compared to the control group. Immunohistochemical study of the skin indicated that following sivelestat treatment, the quantity of CD3+cells in the skin was 1.8-2.2 times lower, and the level of proliferative activity (Ki-67+cells) was 2.3-2.9 lower compared to the non-therapy group. In contrast to topical corticosteroids where the more pronounced anti-inflammatory effect is typically seen with ointment formulations, with sivelestat we observed an opposite effect. The reasons for that reversal remain unclear. Conclusion: Based on the results obtained using the animal model of imiquimod-induced psoriasis, it was established that the HNE inhibitor sivelestat demonstrated efficacy comparable to that of a strong topical glucocorticoid steroidal drug (betamethasone dipropionate 1%). Significant resolution of skin lesions, reduction of epidermal thickness, diminishing of the skin infiltration with T-lymphocytes and normalization of the cell division rate in epidermis and dermis were evident. Thus, suppression of IL-36 mediated inflammation activity in the skin by topical application of a HNE inhibitor represents a promising new direction in the treatment of psoriasis. Certainly, HNE has other targets; thus, molecular studies could be subject of future experiments beyond the scope of the present study.

Inhibition of Neutrophil Elastase and Cathepsin G As a New Approach to the Treatment of Psoriasis: From Fundamental Biology to Development of New Target-Specific Drugs.

Psoriasis therapy remains an extremely relevant area of modern drug design, due to necessity of adverse reaction reduction, inherent for actual methods of therapy. It was established that two serine proteases-neutrophil elastase 1 (HNE1) and cathepsin G (CatG)-are the key agents in psoriasis development. The collected molecular data for the presented targets form the basis for the molecular modeling strategy for the search for and identification of new target-specific inhibitors. The result of this work is a group of high-priority small-molecule compounds with double-targeted affinity, which are able to suppress the pro-psoriatic processes induced by the considered serine proteases at the initial stage of the disease.

Discovery of Novel Isatin-Based p53 Inducers.

A series of isatin Schiff base derivatives were identified during in silico screening of the small molecule library for novel activators of p53. The compounds selected based on molecular docking results were further validated by a high-content screening assay using U2OS human osteosarcoma cells with an integrated EGFP-expressing p53-dependent reporter. The hit compounds activated and stabilized p53, as shown by Western blotting, at higher rates than the well-known positive control Nutlin-3. Thus, the p53-activating compounds identified by this approach represent useful molecular probes for various cancer studies.

[The role of mitochondrial dynamics in cell death].

Mitochondria are dynamic organelles whose homeostasis is defined by two opposite processes: fission (or fragmentation), or fusion. Fission of mitochondria results in generation of smaller organelles and fusion is when they produce tubular or net-like structures. Although a number of proteins are already known to control the process of fission/fusion additional regulators controlling these processes are being found. The Bcl-2 family members take part in the regulation of apoptosis and according to the current view are involved in the mitochondrial net-like structure maintenance. In this review we will discuss mechanisms of mitochondrial fission/fusion regulation and summarize the available information on the role of Bcl-2 family members in the regulation of mitochondrial fission/fusion dynamics.

AMP-activated protein kinase: structure, function, and role in pathological processes.

Recently, AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy balance at cellular and whole-body levels. Due to the involvement in multiple signaling pathways, AMPK efficiently controls ATP-consuming/ATP-generating processes to maintain energy homeostasis under stress conditions. Loss of the kinase activity or attenuation of its expression leads to a variety of metabolic disorders and increases cancer risk. In this review, we discuss recent findings on the structure of AMPK, its activation mechanisms, as well as the consequences of its targets in regulation of metabolism. Particular attention is given to low-molecular-weight compounds that activate or inhibit AMPK; the perspective of therapeutic use of such modulators in treatment of several common diseases is discussed.

Spectral study of the interaction of DNA with benzothiazolyl-benz-alpha-chromene.

Absorption and luminescence excitation and emission spectra of newly synthesized 2-(4-methylphenylimino)-3-(2 -benzothiazolyl)benz-alpha-chromene (BCBT) have been studied in the presence of various DNA concentrations. BCBT is characterized by the existence of two different fluorescent systems, exhibiting radiationless fluorescence resonance energy transfer between them. In the range of molar ratios of polynucleotide/dye concentrations from 0 to 50, BCBT preferentially intercalates into DNA due to its benz-alpha-chromene fragment, whereas the 2-benzothiazolyl fragment is responsible for fluorescence.

Spectral study of interactions of 4,8,4'-trimethylpsoralen and 4,4'-dimethylangelicin dyes with DNA.

Absorption and fluorescence spectra for six new synthetic dyes of 4,8,4'-trimethylpsoralen and 4,4'-dimethylangelicin derivatives containing various terminal substituents at 5'-position have been investigated in different environments using a wide range of the DNA/ligand concentrations. Various spectral and binding characteristics of the DNA-ligand systems have been determined. General principles characterizing mechanisms responsible for changes in the fluorescent properties of nucleotide-specific dyes have been proposed; they take into consideration chemical structure of the dyes, properties of the environment, and degree of sorption on substrate.

[Comparative study of DNA-specific dyes of the indole and benzimidazole derivates]. / Sravnitel'noe issledovanie svoistv DNK-spetsifichnykh krasitelei indol'nogo i benzimidazol'nogo riada.

Various models of complex formation of low-molecular ligands with DNA are considered. Using the Scatchard model, parameters of binding of fluorescent monophenylindole, monophenylbenzimidazole, and bisbenzimidazole dyes with calf thymus DNA were evaluated. By means of graphic (nonparametric) and correlation analysis, various spectral and complexation properties of these dyes in the presence of DNA are compared.

[Spectral properties of bisbenzimidazole dyes upon interaction with DNA]. / Spektral'nye svoistva krasitelei bisbenzimidazol'nogo riada pri vzaimodeistvii s DNK.

Absorption and fluorescence spectra of a series of bisbenzimidazole dyes were studied in the presence of various concentrations of DNA and in diverse media. Variations in spectral properties of the compounds under study were found to depend on the measurement conditions. The bases for selecting the optimal composition of the media for DNA detection were presented. The modes of substrate-ligand binding and the dynamics of the alterations of fluorescent properties of structurally varying dyes upon their interaction with DNA were considered.

[Mechanisms for changing fluorescent properties of bis-benzimidazole dyes]. / Mekhanizmy izmeneniia fluorestsentnykh svoistv bis-benzimidazol'nykh krasitelei.

Spectral properties of a series of externally binding substrate-specific bis-benzimidazole dyes that were either bound or not bound to DNA were studied. Feasible mechanisms of variation in fluorescent properties of the dyes under study were considered depending on their chemical structure. Contribution of the rotational diffusion and the donor-acceptor interactions to the prediction and explanation of fluorescent properties of the substrate-specific dyes was discussed.

[Fluorescence characteristics of DNA complexes with dyes of the bis-benzimidazole series]. / Issledovanie fluorestsentnykh kharakteristik kompleksov s DNK krasitelei riada bis-benzimidazolov.

Fluorescence characteristics of DNA-specific dyes of bis-benzimidazole type in a wide range of pH and r = C/P were investigated. Fluorescence spectra of DNA complexes with bis-benzimidazoles have elements of a structure, which may result from a superposition of the spectra of dye molecules in different protonization group states that form different types of complexes with DNA. Experimental data do not contradict the idea of bis-benzimidazole dye binding into the minor groove of DNA. Bis-benzimide molecules in the deprotonization state have a major affinity to DNA.