The application of machine learning methods to the prediction of novel ligands for RORγ/RORγT receptors.

In this work, we developed and applied a computational procedure for creating and validating predictive models capable of estimating the biological activity of ligands. The combination of modern machine learning methods, experimental data, and the appropriate setup of molecular descriptors led to a set of well-performing models. We thoroughly inspected both the methodological space and various possibilities for creating a chemical feature space. The resulting models were applied to the virtual screening of the ZINC20 database to identify new, biologically active ligands of RORγ receptors, which are a subfamily of nuclear receptors. Based on the known ligands of RORγ, we selected candidates and calculate their predicted activities with the best-performing models. We chose two candidates that were experimentally verified. One of these candidates was confirmed to induce the biological activity of the RORγ receptors, which we consider proof of the efficacy of the proposed methodology.

RORγT agonists as immune modulators in anticancer therapy.

RORγT is a transcription factor that directs the development of Th17 lymphocytes and other IL-17-expressing cells (e.g., Tc17 and ILC3 cells). These cells are involved in the body's defense against pathogenic bacteria and fungi, but they also participate in maintaining the proinflammatory environment in some autoimmune diseases and play a role in the immune system's response to cancer. Similar to other members of the nuclear receptor superfamily, the activity of RORγT is regulated by low-molecular-weight ligands. Therefore, extensive efforts have been dedicated to identifying inverse agonists that diminish the activity of this receptor and subsequently inhibit the development of autoimmune diseases. Unfortunately, in the pursuit of an ideal inverse agonist, the development of agonists has been overlooked. It is important to remember that these types of compounds, by stimulating lymphocytes expressing RORγT (Th17 and Tc17), can enhance the immune system's response to tumors. In this review, we present recent advancements in the biology of RORγT agonists and their potential application in anticancer therapy.

Anti-hepatocellular carcinoma activity of the cyclin-dependent kinase inhibitor AT7519.

Hepatocellular carcinoma (HCC) is one of the most common cancerous tumors and one of the leading causes of death among cancer-related disorders. Chemotherapy is ineffective in HCC patients, and the number of drugs that are in use is limited. Thus, new molecules are needed that could increase the effectiveness of anti-HCC regimens. Here, we show that AT7519, a CDK inhibitor, exerts positive effects on HCC cells: it inhibits proliferation, migration and clonogenicity. Detailed analysis of the transcriptomes of cells treated with this compound indicated that AT7519 affects a substantial portion of genes that are associated with HCC development and progression. Moreover, we showed that the concomitant use of AT7519 with gefitinib or cabozantinib sensitized HCC cells to these drugs. Thus, our research indicates that AT7519 is worth considering in monotherapy for hepatocellular carcinoma patients or in combination with other drugs, e.g., gefitinib or cabozantinib.

Trichostatin A inhibits expression of the human SLC2A5 gene via SNAI1/SNAI2 transcription factors and sensitizes colon cancer cells to platinum compounds.

GLUT5, a key protein encoded by the SLC2A5 gene, is involved in the uptake of fructose from the intestine. Currently, with the increased consumption of this sugar and the associated increased incidence of obesity, diabetes and cancer, GLUT5 may represent an important molecular target in the prevention and treatment of these diseases. Here, we demonstrate that overexpression of the SNAI1 and SNAI2 transcription factors in cells expressing high levels of SLC2A5 mRNA reduced SLC2A5 gene expression. Furthermore, a histone deacetylase inhibitor, trichostatin A, which induces SNAI1 and SNAI2 expression, inhibits SLC2A5/GLUT5 expression and sensitizes colon cancer cells to cisplatin and oxaliplatin. This finding might have potential relevance for the development of therapeutic treatments aimed at modulating fructose transport or genes involved in this process for use with certain cancers.

Chlorpromazine, a Clinically Approved Drug, Inhibits SARS-CoV-2 Nucleocapsid-Mediated Induction of IL-6 in Human Monocytes.

The COVID-19 pandemic, caused by the rapidly spreading SARS-CoV-2 virus, led to the unprecedented mobilization of scientists, resulting in the rapid development of vaccines and potential pharmaceuticals. Although COVID-19 symptoms are moderately severe in most people, in some cases the disease can result in pneumonia and acute respiratory failure as well as can be fatal. The severe course of COVID-19 is associated with a hyperinflammatory state called a cytokine storm. One of the key cytokines creating a proinflammatory environment is IL-6, which is secreted mainly by monocytes and macrophages. Therefore, this cytokine has become a target for some therapies that inhibit its biological action; however, these therapies are expensive, and their availability is limited in poorer countries. Thus, new cheaper drugs that can overcome the severe infections of COVID-19 are needed. Here, we show that chlorpromazine inhibits the expression and secretion of IL-6 by monocytes activated by SARS-CoV-2 virus nucleocapsid protein and affects the activity of NF-κB and MEK/ERK signaling. Our results, including others, indicate that chlorpromazine, which has been used for several decades as a neuroleptic, exerts antiviral and immunomodulatory activity, is safe and inexpensive, and might be a desirable drug to support the therapy of patients with COVID-19.

Targeting EGFR in melanoma - The sea of possibilities to overcome drug resistance.

Melanoma is considered one of the most aggressive skin cancers. It spreads and metastasizes quickly and is intrinsically resistant to most conventional chemotherapeutics, thereby presenting a challenge to researchers and clinicians searching for effective therapeutic strategies to treat patients with melanoma. The use of inhibitors of mutated serine/threonine-protein kinase B-RAF (BRAF), e.g., vemurafenib and dabrafenib, has revolutionized melanoma chemotherapy. Unfortunately, the response to these drugs lasts a limited time due to the development of acquired resistance. One of the proteins responsible for this process is epidermal growth factor receptor (EGFR). In this review, we summarize the role of EGFR signaling in the multidrug resistance of melanomas and discuss possible applications of EGFR inhibitors to overcome the development of drug resistance in melanoma cells during therapy.

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

Hypoxia modulates human mast cell adhesion to hyaluronic acid.

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

Do Mast Cells Contribute to the Antifungal Host Defense?

The fungal kingdom includes a group of microorganisms that are widely distributed in the environment, and therefore the exposure to them is almost constant. Furthermore, fungal components of the microbiome, i.e., mycobiome, could serve as a reservoir of potentially opportunistic pathogens. Despite close encounters with fungi, defense mechanisms that develop during fungal infections remain unexplored. The strategic location of mast cells (MCs) close to the external environment places them among the first cells to encounter pathogens along with the other innate immune cells. MCs are directly involved in the host defense through the ability to destroy pathogens or indirectly by activating other immune cells. Most available data present MCs' involvement in antibacterial, antiviral, or antiparasitic defense mechanisms. However, less is known about their contribution in defense mechanisms against fungi. MCs may support immune responses to fungi or their specific molecules through initiated degranulation, synthesis and release of cytokines, chemokines, mediators, and generation of reactive oxygen species (ROS), as well as immune cells' recruitment, phagocytosis, or provision of extracellular DNA traps. This review summarizes current knowledge on host defense mechanisms against fungi and MCs' involvement in those processes. It also describes the effects of fungi or fungus-derived constituents on MCs' activity.

The Art of Mast Cell Adhesion.

Cell adhesion is one of the basic phenomena occurring in a living organism, affecting many other processes such as proliferation, differentiation, migration, or cell viability. Mast cells (MCs) are important elements involved in defending the host against various pathogens and regulating inflammatory processes. Due to numerous mediators, they are contributing to the modulation of many basic cellular processes in a variety of cells, including the expression and functioning of different adhesive molecules. They also express themselves many adhesive proteins, including ICAM-1, ICAM-3, VCAM-1, integrins, L-selectin, E-cadherin, and N-cadherin. These molecules enable MCs to interact with other cells and components of the extracellular matrix (ECM), creating structures such as adherens junctions and focal adhesion sites, and triggering a signaling cascade. A thorough understanding of these cellular mechanisms can create a better understanding of MC biology and reveal new goals for MC targeted therapy. This review will focus on the current knowledge of adhesion mechanisms with the involvement of MCs. It also provides insight into the influence of MCs or MC-derived mediators on the adhesion molecule expression in different cells.

Hypoxia regulates human mast cell adhesion to fibronectin via the PI3K/AKT signaling pathway.

A decrease in oxygen concentration is a hallmark of inflammatory reactions resulting from infection or homeostasis disorders. Mast cells interact with extracellular matrix and other cells by adhesion receptors. We investigated the effect of hypoxia on integrin-mediated mast cell adhesion to fibronectin. We found that it was mediated by the α5/ß1 receptor and that hypoxia significantly upregulated this process. Hypoxia-mediated increases in mast cell adhesion occurred without increased surface expression of integrins, suggesting regulation by inside-out integrin signaling. Hypoxia also mediated an increase in phosphorylation of Akt, and PI3'kinase inhibitors abolished hypoxia-mediated mast cell adhesion. Hypoxia upregulates the function of integrin receptors by PI3' kinase-dependent signaling. This process might be important for the location of mast cells at inflammatory sites.

Adipocytokine Involvement in Innate Immune Mechanisms.

The innate immune response is defined as an immensely complex and sophisticated process aimed at defending the organism against any disturbance in the body homeostasis, including invading pathogens. It requires a close cooperation of a vast amount of different cell types, recognized as inflammatory migrating cells, as well as stationary cells that form tissues. Moreover, innate immune mechanisms require an efficient functioning of various humoral components that exert a significant impact on physiological and pathological processes. Apart from commonly mentioned humoral factors, this group also includes a family of proteins known as adipocytokines that may act as pro- or anti-inflammatory agents or act both ways. Leptin, predominantly characterized as a proinflammatory adipokine, plays a crucial role in endothelium remodeling and regulation, as well as in cell survival and production of numerous cytokines. Adiponectin, similar to leptin, acts on the endothelial cells and the phagocytic properties of immune cells; however, it exerts an anti-inflammatory impact. Resistin has a documented role in the control of angiogenesis and stimulation of proinflammatory mediator generation and release. Furthermore, there are adipokines, ie, visfatin and chemerin, whose participation in the inflammatory processes is ambiguous. This review focuses on the current knowledge on the extensive role of selected adipokines in innate immune response.

The RLR/NLR expression and pro-inflammatory activity of tissue mast cells are regulated by cathelicidin LL-37 and defensin hBD-2.

Considering the significance of mast cells (MCs) in the course of various physiological and pathological processes, and the pivotal role of endogenous molecules, i.e., cathelicidins and defensins as multifunctional modulators, the study examines the constitutive and cathelicidin LL-37/defensin hBD-2-induced expression of certain NLRs and RLRs, i.e., NOD1, NOD2, and RIG-I, in fully-mature tissue MCs, and the impact of LL-37 and hBD-2 on MC pro-inflammatory activity. All experiments were carried out in vitro on freshly-isolated peritoneal (P)MCs. qRT-PCR, western blotting, flow cytometry, and confocal microscopy were used to evaluate both constitutive and LL-37/hBD-2-induced expression of NOD1, NOD2, and RIG-I receptors. ROS was determined using H2DCFDA, and Boyden microchamber assay was used to define the migratory response. Standard techniques assessed histamine, cysLT, and chemokine generation. PMCs express NOD1, NOD2, and RIG-I constitutively. LL-37 and hBD-2 enhance the expression and induce translocation of the studied receptors and directly activate the pro-inflammatory and migratory responses of PMCs. Observations demonstrate that LL-37 and hBD-2 might augment MC capability and sensitivity to NLR and RLR ligands and strengthen the role of MCs in inflammation.

An overview of mast cell pattern recognition receptors.

BACKGROUND: Mast cells (MCs) are long-lived immune cells of the connective tissue which play a key role in development and amplification of inflammatory process initiated inter alia by allergic reactions or microbial infections. They reside in strategic locations in the body that are notably exposed to deleterious factors disturbing homeostasis, which enables them to become one of the first-line defense strategy. MCs have developed a wide range of various mechanisms to deal with invading intruders and harmful endogenic factors. Those include storage and synthesis with a subsequent release of inflammatory mediators, forming of MC-extracellular traps, and phagocytosis. FINDINGS: Particularly, important role in microbial sensing is achieved due to the presence of different pattern recognition receptors (PRRs). The best-described receptors are Toll-like receptors activated by different pathogen- and damage-associated molecular patterns. However, MCs express also C-type lectin receptors specialized in antifungal defense, NOD-like receptors detecting bacterial peptidoglycans, and RIG-like receptors relevant in viral sensing. CONCLUSION: This review will focus on the current knowledge of PRRs expressed within different types of MCs.

Mast cells as the strength of the inflammatory process.

The inflammatory process is a complex host defence mechanism aimed at the elimination of deleterious factors disturbing homeostasis. Inflammation consists of several interdependent stages controlled by a wide range of mediators. Those include acute phase proteins, heat shock proteins, complement components, biogenic amines, cytokines, lipid-derived mediators, reactive oxygen species, nitric oxide, proteolytic enzymes, and kinins. Due to the strategic location in the body, mast cells play a protective role in the inflammatory process, through its initiation, amplification, and resolution. Mast cells degranulate and/or newly produce, and release various mediators classified into three groups: preformed mediators, de novo synthesised lipid mediators, and newly synthesised cytokines. Those mediators have an impact on different processes occurring during inflammation, inter alia, they influence blood vessels leading to dilation, enhanced adhesion molecule expression, and increased permeability. Furthermore, mast cell mediators play a pivotal role in inflammatory cell chemotaxis, degradation of extracellular matrix proteins, impact on stationery cells and resolution of inflammation. The release of mast cell mediators and their actions constitute a highly complex and still not fully understood mechanism, which warrants further studies of the action of mast cells in inflammation. This review will focus on the current knowledge concerning the broad role of mast cells in the inflammatory process.