Synthesis and multifaceted pharmacological activity of novel quinazoline NHE-1 inhibitors

The Na+/H+ exchanger isoform 1 (NHE-1) attracts ongoing attention as a validated drug target for the management of cardiovascular and ocular diseases owing to cytoprotective, anti-ischemic and anti-inflammatory properties of NHE-1 inhibitors. Herein we report novel NHE-1 inhibitors realized via functionalization of N1-alkyl quinazoline-2,4(1H,3H)-dione and quinazoline-4(3H)-one with N-acylguanidine or 3-acyl(5-amino-1,2,4-triazole) side chain. Lead compounds show activity in a nanomolar range. Their pharmacophoric features were elucidated with neural network modeling. Several compounds combine NHE-1 inhibition with antiplatelet activity. Compound 6b reduces intraocular pressure in rats and effectively inhibits the formation of glycated proteins. Compounds 3e and 3i inhibit pro-inflammatory activation of murine macrophages, LPS-induced interleukin-6 secretion and also exhibit antidepressant activity similar to amiloride. Hence, novel compounds represent an interesting starting point for the development of agents against cardiovascular diseases, thrombotic events, excessive inflammation, long-term diabetic complications and glaucoma.

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

Synthesis And Multifaceted Pharmacological Activity of Novel Quinazoline NHE-1 Inhibitors

The Na+/H+ exchanger isoform 1 (NHE-1) attracts ongoing attention as a validated drug target for the management of cardiovascular and ocular diseases owing to cytoprotective, anti-ischemic and anti-inflammatory properties of NHE-1 inhibitors. Herein we report novel NHE-1 inhibitors realized via functionalization of N1-alkyl quinazoline-2,4(1H,3H)-dione and quinazoline-4(3H)-one with N-acylguanidine or 3-acyl(5-amino-1,2,4-triazole) side chain. Lead compounds show activity in a nanomolar range. Their pharmacophoric features were elucidated with neural network modeling. Several compounds combine NHE-1 inhibition with antiplatelet activity. Compound 6b reduces intraocular pressure in rats and effectively inhibits the formation of glycated proteins. Compounds 3e and 3i inhibit pro-inflammatory activation of murine macrophages, LPS-induced interleukin-6 secretion and also exhibit antidepressant activity similar to amiloride. Hence, novel compounds represent an interesting starting point for the development of agents against cardiovascular diseases, thrombotic events, excessive inflammation, long-term diabetic complications and glaucoma.

Proteomic Investigation of Glyceraldehyde-Derived Intracellular AGEs and Their Potential Influence on Pancreatic Ductal Cells

Glyceraldehyde-derived advanced glycation end products (AGEs) play an important role in the pathogenesis of many diseases including cancer. Accumulation of intracellular AGEs could stimulate cancer induction and facilitate cancer progression. We evaluated the toxic effect of glyceraldehyde-derived intracellular AGEs on normal and malignant pancreatic ductal cells by assessing the cell viability, toxicity, and oxidative stress, followed by proteomic analysis. Our functional studies showed that pancreatic cancer cells (PANC-1 and MIA PaCa-2) were more resistant to glyceraldehyde treatment compared to normal pancreatic ductal epithelial cells (HPDE), while cytotoxicity effects were observed in all cell types. Furthermore, using 13C isotopic labeled glyceraldehyde, the proteomic data revealed a dose-dependent increment of the number of glycation adducts in both these cell types. HPDE cells showed a higher number of intracellular AGEs compared to cancer cells. At a molecular level, the glycations in the lysine residues of proteins showed a concurrent increase with the concentration of the glyceraldehyde treatment, while the arginine glycations appeared to be less affected by the glyceraldehyde doses. Further pathway analysis of these glycated proteins suggested that the glycated proteins participate in important biological processes that are major hallmarks of cancer initiation and progression, including metabolic processes, immune response, oxidative stress, apoptosis, and S100 protein binding.

Effect of enzymatic glycosylation on the structure and properties of wheat gluten protein fibers

Wheat gluten proteins are good raw materials for preparing fibers due to their excellent viscoelasticity. However, protein fibers made directly from wheat gluten have poor mechanical properties. In this paper, transglutaminase was used to induce the glycosylation reaction between wheat gluten proteins and carboxymethyl chitosan. The glycated proteins were then made into fibers by wet spinning. After glycosylation modification, the breaking strength and breaking elongation of the wheat gluten protein fibers (WGPF) improved by 43% and 127%, respectively. Fourier transform infrared spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses revealed that the glycosylation-modified WGPF molecules contained saccharide portions, which confirms the covalent attachment of carboxymethyl chitosan to the wheat gluten protein. Scanning electron microscopy showed that the number of pores in the cross-section of the modified WGPF was lower than that in the unmodified WGPF. The thermal stability and dyeability of the modified WGPF were also improved.

Physiological and pathogenic role of scavenger receptors in humans

The scavenger receptors (SRs)) include > 30 different molecules structurally classified into 11 classes (A to L). They are expressed mostly on stromal macrophages, and their expression may be augmented in direct dependence with concentrations of their ligands. The SRs are heterogenous by their structure, however, being common in their functional potential. E.g., different SR classes may participate in absorption of modified low-density lipoproteins and glycated proteins, apoptotic and ageing cells, altered erythrocytes and platelets, like as a big variety of other endogenous ligands from metabolic and cellular “trash”. A common property of SRs is their participation in removal of small pathogen amounts from blood circulation, regulation of cell and tissue stress responses, ability to form complicated receptor complexes with other receptor types including integrins and toll-like receptors. Opposite to classic pattern-recognizing receptors, the SR involvement does not always elicit a pronounced cellular activation and development of pro-inflammatory cellular stress. The SR functional effects provide interactions between different physiological events and immune system, including the processes of neuroendocrine and metabolic regulation. These mechanisms provide both homeostatic stability and, likewise, act at the border of normal and pathological conditions, i.e., participating in pathogenesis of transitional processes, e.g., physiological ageing. Moreover, the SR-associated processes represent a key pathogenetic factor in different somatic diseases, e.g., those associated with low-intensity chronic inflammation, including obesity, type 2 diabetes, atherosclerosis, arterial hypertension, various neurodegenerative disorders. Similarly, the SRs are involved into the processes of cancer transformation and antitumor response, different processes of classical inflammation, from antigen presentation to the morphofunctional T cell and macrophage polarization in the inflammation foci and immunocompetent organs. SR are playing a controversial role in development of acute systemic inflammation, the main reason for lethal outcomes in the intensive care wards. Targeted effects upon the SRs represent a promising approach when treating a broad variety of diseases, whereas detection of membrane-bound and soluble SR forms could be performed by means of diagnostic and monitoring techniques in many human disorders.

Glycated proteins

Glycation is a biological reaction that occurs in all proteins. Thisreaction proceeds more slowly in healthy subjects and more rapidly in patients suffering from a hyperglycemia. Glycated proteins cannot fulfill their functions that could lead to metabolic disorders. The process of glycation leads to building of advanced glycation end-products (AGEs). Thestructureof AGEs has not been fully researched yet. Glycated proteins have diagnostic meaning in different health conditions and not only in patients with diabetes mellitus. Determination of glycated proteins level (hemoglobin and plasma proteins) in diagnostics of diabetes mellitus and the effectiveness of its treatment; measurements of glycated proteins could be used as a predictor of different illnesses and their complications. Glycated hemoglobin was researched in children with diabetes mellitus of different severity. It has been shown that the level of glycated proteins does not always correlate with blood sugar level. Results of glycated proteins measurements in patients with thyroid disorders shows that the glycation takes place not only in patients with diabetes mellitus, but also with other illnesses without hyperglycemia. Our research in patients with diabetes mellitus has shown that the measured level of glycated proteins and plasma proteins could be more significant in the course of disease than the level of blood sugar. Compensation of diabetes mellitus in children in regard of the blood sugar level does not always correlate with the level of glycated proteins. This assumption could lead to the conclusion that only the combination of measurements like blood sugar, glycated hemoglobin and glycated proteins could give a full picture of disease compensation.

Association of Glycated Proteins with Inflammatory Proteins and Periodontal Disease Parameters

Periodontitis is a chronic inflammatory condition that may contribute to diabetogenesis. The aim was to investigate the levels of glycated proteins and their correlation with periodontal and systemic inflammation. Fifty-one patients with periodontitis and 20 healthy subjects underwent probing pocket depth (PPD) measurements. PPD total and PPD disease with and without tooth adjustment were used as continuous indices. Marginal bone loss (MBL) for mandibular premolars and molars was measured digitally. Body mass index (BMI) and waist circumference (WC) were also analyzed. Glycated hemoglobin (HbA1c) and fructosamine (FrAm) levels were measured in all subjects. A multiplex proximity extension assay (PEA) was used to analyze the serum samples for simultaneous measurement of 92 proteins. Both HbA1c and FrAm inversely correlated with IL-10, FGF-21, MCP-1, and TNF beta amongst 16 proteins. HbA1c correlated directly with OPG. Parameters of disease severity were consistently significant for HbA1c. Adjusted PPD total and number of missing teeth were increased in diabetes whereas levels of RANKL and RANKL to OPG ratio were the highest in nondiabetic periodontitis patients. Hyperglycemic conditions in periodontitis patients are associated with reduced levels of anti-inflammatory proteins as well as dysregulated bone resorption.