Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo.

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

The impact of selected xanthophylls on oil hydrolysis by pancreatic lipase: in silico and in vitro studies.

Lipase inhibition is one of the directions to control obesity. In vitro assays have confirmed the inhibitory effect of selected xanthophylls, including astaxanthin, fucoxanthinol, fucoxanthin, and neoxanthin. Similarly, an in-silico study also demonstrated the successful inhibition of pancreatic lipase by astaxanthin. Unfortunately, the efficacy of these protocols in the emulsion state typical of lipid digestion remains untested. To address this issue, the current study employed the pH-stat test, which mimics lipid digestion in the gastrointestinal tract, to evaluate native and prepared sea buckthorn and rapeseed oils with varying xanthophyll contents from 0 to 1400 mg/kg oil. Furthermore, a molecular docking of zeaxanthin and violaxanthin (commonly found in plant-based foods), astaxanthin (widely distributed in foods of marine origin) and orlistat (approved as a drug) was performed. The in-silico studies revealed comparable inhibitory potential of all tested xanthophylls (variation from - 8.0 to - 9.3 kcal/mol), surpassing that of orlistat (- 6.5 kcal/mol). Nonetheless, when tested in an emulsified state, the results of pH-stat digestion failed to establish the inhibitory effect of xanthophylls in the digested oils. In fact, lipolysis of native xanthophyll-rich sea buckthorn oil was approximately 22% higher than that of the xanthophyll-low preparation. The key insight derived from this study is that the amphiphilic properties of xanthophylls during the digestion of xanthophyll-rich lipids/meals facilitate emulsion formation, which leads to enhanced fat lipolysis.

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization.

Protein prenylation is a post-translational modification controlling the localization, activity, and protein-protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.

Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase.

Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of eukaryotic Rab GTPases, proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of Rab GTPases. Here, a series of new α-phosphonocarboxylates have been prepared in the first attempt of rational design of covalent inhibitors of RGGT derived from non-covalent inhibitors. These compounds were equipped with electrophilic groups capable of binding cysteines, which are present in the catalytic cavity of RGGT. A few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line. The proposed mechanism of this inhibitory activity was rationalised by molecular docking and mass spectrometric measurements, supported by stability and reactivity studies.

Composition of flesh lipids and oleosome yield optimization of selected sea buckthorn (Hippophae rhamnoides L.) cultivars grown in Poland.

Sea buckthorn berries contain lipids rich in palmitoleic acid, carotenoids, tocols and sterols, but their composition varies greatly depending on the cultivar and region of cultivation. Therefore, the current study presents the chemical composition of fruit flesh oils of cultivars grown in Poland and compares them with plants grown worldwide. Among tested cultivars, the highest shares of palmitoleic acid were determined in Golden Rain and Luczystaja cvs. Ten grams of sea buckthorn flesh oil provides at least 28% of vitamin A, 50% of vitamin E and 5% of sterols of the recommended dietary allowance (RDA) values for adults. The final part of this study is dedicated to a preliminary study of the optimization of the oleosome yield by the centrifugation method. The maximum oleosome yield can be obtained at a relatively low centrifugal force (below 8000×g), while optimal temperature and time should be laboratory determined for each cultivar.

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts, Keratinocytes, and Endothelial Cells.

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5'-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5'-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase.

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

Stable composite of bacterial nanocellulose and perforated polypropylene mesh for biomedical applications.

The article presents the method of preparation of new, stable bacterial cellulose composites with perforated solid materials for biomedical applications, comprising reconstructive surgery of soft and hard tissues. The composites were obtained in specially designed bioreactors equipped with a set of perforated mesh stripes threaded vertically to the culture medium, ensuring perpendicular growth of bacterial nanocellulose synthesized by Komagataeibacter xylinus E25 in stationary culture. The developed biocomposites have been tested for stability and mechanical strength, as well as for their in vitro inflammatory responses shown as mast cell degranulation with N-acetyl-ß-d-hexosaminidase release and mast cell adhesion. The obtained results indicate that the composites components are well integrated after the process of cultivation and purification. Bacterial nanocellulose does not negatively influence mechanical properties of the polypropylene porous mesh, preserving its tensile strength, elasticity, and load. Moreover, application of bacterial cellulose makes the composites less immunogenic as compared to polypropylene itself. Therefore, the composites have the great potential of application in medicine, and depending on the applied porous material, might be used either in hernioplasty (if porous hernia mesh is used), cranioplasty (if perforated metal or polymeric cranial implant is applied), or as a protective barrier in any application that requires biocompatibility or antiadhesive properties improvement. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 978-987, 2019.

Lysophosphatidylcholine and its phosphorothioate analogues potentiate insulin secretion via GPR40 (FFAR1), GPR55 and GPR119 receptors in a different manner.

Lysophosphatidylcholine (LPC) is an endogenous ligand for GPR119 receptor, mediating glucose-stimulated insulin secretion (GSIS). We demonstrate that LPC facilitates GSIS in MIN6 pancreatic ß-cell line and murine islets of Langerhans by recognizing not only GPR119 but also GPR40 (free fatty acid receptor 1) and GPR55 activated by lysophosphatidylinositol. Natural LPCs are unstable when administered in vivo limiting their therapeutic value and therefore, we present phosphorothioate LPC analogues with increased stability. All the modified LPCs under study (12:0, 14:0, 16:0, 18:0, and 18:1) significantly enhanced GSIS. The 16:0 sulfur analogue was the most potent, evoking 2-fold accentuated GSIS compared to the native counterpart. Interestingly, LPC analogues evoked GPR40-, GPR55-and GPR119-dependent [Ca2+]i signaling, but did not stimulate cAMP accumulation as in the case of unmodified molecules. Thus, introduction of a phosphorothioate function not only increases LPC stability but also modulates affinity towards receptor targets and evokes different signaling pathways.

Identification of the Privileged Position in the Imidazo[1,2-a]pyridine Ring of Phosphonocarboxylates for Development of Rab Geranylgeranyl Transferase (RGGT) Inhibitors.

Members of the Rab GTPase family are master regulators of vesicle trafficking. When disregulated, they are associated with a number of pathological states. The inhibition of RGGT, an enzyme responsible for post-translational geranylgeranylation of Rab GTPases represents one way to control the activity of these proteins. Because the number of molecules modulating RGGT is limited, we combined molecular modeling with biological assays to ascertain how modifications of phosphonocarboxylates, the first reported RGGT inhibitors, rationally improve understanding of their structure-activity relationship. We have identified the privileged position in the core scaffold of the imidazo[1,2-a]pyridine ring, which can be modified without compromising compounds' potency. Thus modified compounds are micromolar inhibitors of Rab11A prenylation, simultaneously being inactive against Rap1A/Rap1B modification, with the ability to inhibit proliferation of the HeLa cancer cell line. These findings were rationalized by molecular docking, which recognized interaction of phosphonic and carboxylic groups as decisive in phosphonocarboxylate localization in the RGGT binding site.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner.

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

Nucleoside 5'-O-monophosphorothioates as modulators of the P2Y14 receptor and mast cell degranulation.

Mast cells (MCs) are long-lived resident cells known for their substantial role in antigen-induced anaphylaxis and other immunoglobulin E-mediated allergic reactions as well as tumor promotion. MCs' activation results in the release of pro-inflammatory factors such as histamine, tryptase, tumor necrosis factor or carboxypeptidase A stored in secretory granules. IgE-dependent hypersensitivity has been thought to be the major pathway mediating degranulation of mast cells, but the P2Y14 nucleotide receptor activated by UDP-glucose (UDPG) may also enhance this process. In this study we identified thymidine 5'-O-monophosphorothioate (TMPS) as a molecule inhibiting UDPG-induced degranulation in a rat mast cell line (RBL-2H3). Additionally, TMPS diminished UDPG-evoked intracellular calcium mobilization in a stable HEK293T cell line overexpressing the P2Y14 receptor. Therefore, we demonstrate that the use of thymidine 5'-O-monophosphorothioate might be a novel anti-inflammatory approach based on preventingmast cell activation.

The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine.

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

Thymidine 5'-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor.

ATP, ADP, UTP, and UDP acting as ligands of specific P2Y receptors activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, migration, differentiation, and cell death. Contrary to a widely held opinion, we show here that nucleoside 5'-O-monophosphorothioate analogs, containing a sulfur atom in a place of one nonbridging oxygen atom in a phosphate group, act as ligands for selected P2Y subtypes. We pay particular attention to the unique activity of thymidine 5'-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor (P2Y6R). We also collected evidence for the involvement of the P2Y6 receptor in human epithelial adenocarcinoma cell line (HeLa) cell migration induced by thymidine 5'-O-monophosphorothioate analog. The stimulatory effect of TMPS was abolished by siRNA-mediated P2Y6 knockdown and diisothiocyanate derivative MRS 2578, a selective antagonist of the P2Y6R. Our results indicate for the first time that increased stability of thymidine 5'-O-monophosphorothioate as well as its affinity toward the P2Y6R may be responsible for some long-term effects mediated by this receptor.

Proangiogenic properties of nucleoside 5'-O-phosphorothioate analogues under hyperglycaemic conditions.

Diabetes leads to impairment of the normal course of wound healing. Interestingly, recent studies have implicated a critical role of P2X/P2Y nucleotide receptors in dermal tissue regeneration and maintaining vascular homeostasis. As new vessel generation and keratinization process are decreased in diabetic patients we determined whether nucleoside 5'-O-phosphorothioate analogues might accelerate vascular endothelial growth factor (VEGF) production as well as the growth and migration of human keratinocytes under hyperglycaemic conditions. We also investigated the expression pattern of P2X/P2Y receptors in human keratinocyte HaCaT cells. We show here that nucleoside 5'-Ophosphorothioate analogues are better candidates to overcome hyperglycaemia-induced impairment of angiogenesis as compared to their unmodified counterparts. The greatest potency for VEGF release and stimulation of cell migration by thiophosphate analogues of ATP and UTP correlates with the highest P2Y2 receptor expression by HaCaT cells. We also found that UTPαS significantly increased the viability and proliferation of the HaCaT cells. These findings suggest that thiophosphate analogues of nucleotides could serve as potential therapeutic agents for promoting impaired angiogenesis under diabetic conditions.

Sulfur- and acyl chain-dependent influence of 2-methoxy-lysophosphatidylcholine analogues on ß pancreatic cells.

Nutrient-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is one of the key mechanisms responsible for insulin release from pancreatic islet ß cells. Lysophosphatidylcholine (LPC) was demonstrated to induce insulin secretion from ß cells, activate glucose uptake and effectively lower blood glucose levels in mouse models of type 1 and 2 diabetes mellitus. The article hereby presents the results of a characterization of 2-OMe-LPC sulfur analogues with defined acyl residues in terms of their effect on intracellular Ca(2+) concentration and cellular membrane integrity in the murine ßTC-3 cell model. Active LPC series that could induce calcium flux in ßTC-3 cell model include unmodified LPC 12:0, 14:0, 16:0, and 18:0 as well as phosphorothioate analogues of LPC 12:0, 14:0 and 16:0. However, in the case of species bearing mirystoyl and palmitoyl residues [Ca(2+)]i was associated with membrane permeabilization as demonstrated by propidium iodide incorporation and lactate dehydrogenase release. LPC 12:0 (both unmodified and a sulfurcontaining counterpart) and unmodified LPC 18:0 did not demonstrate membrane disruption but acted as calcium inducers. Interestingly, no stimulation of calcium flux or membrane disruption was observed in the case of LPC analogues with two sulfur atoms introduced into a phosphate group. Experiments with nitrendipine and NiCl2 blocking voltage-dependent calcium channels and the general calcium influx, respectively, revealed remarkably that the compounds studied were involved in different signaling mechanisms while administered to the cell culture, which is clearly related to their chemical structure, both acyl chain and modification dependently.

The chemical synthesis and cytotoxicity of new sulfur analogues of 2-methoxy-lysophosphatidylcholine.

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1 → 2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.

Novel bioactive glycerol-based lysophospholipids: new data -- new insight into their function.

Based on the results of research conducted over last two decades, lysophospholipids (LPLs) were observed to be not only structural components of cellular membranes but also biologically active molecules influencing a broad variety of processes such as carcinogenesis, neurogenesis, immunity, vascular development or regulation of metabolic diseases. With a growing interest in the involvement of extracellular lysophospholipids in both normal physiology and pathology, it has become evident that those small molecules may have therapeutic potential. While lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been studied in detail, other LPLs such as lysophosphatidylglycerol (LPG), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE) or even lysophosphatidylcholine (LPC) have not been elucidated to such a high degree. Although information concerning the latter LPLs is sparse as compared to LPA and S1P, within the last couple of years much progress has been made. Recently published data suggest that these compounds may regulate fundamental cellular activities by modulating multiple molecular targets, e.g. by binding to specific receptors and/or altering the structure and fluidity of lipid rafts. Therefore, the present review is devoted to novel bioactive glycerol-based lysophospholipids and recent findings concerning their functions and possible signaling pathways regulating physiological and pathological processes.

Proangiogenic activity of plant extracts in accelerating wound healing - a new face of old phytomedicines.

Angiogenesis, the formation of new capillaries from pre-existing vascular network, plays an important role in physiological and pathological processes such as embryonic development, wound healing, and development of atherosclerosis. Extension of the circulatory network is also considered to be one the most important factors during cancerogenesis. Inhibition of angiogenesis may lead to inhibition of tumor growth whereas stimulation may improve wound healing. Research achievements suggest the use of plants and their extracts as potential therapeutic agents with pro- or antiangiogenic activity. Since the anticancer and antiangiogenic properties of many phytomedicines have been amply reviewed elsewhere this paper will focus on the treatment of vascular insufficiency in wound healing. Globally accepted herbal drugs are thought to be safe and effective, however, there is a need for more evidence-based confirmation in controlled and validated trials. Among the most frequently studied proangiogenic phytochemicals are ginsenosides from Panax ginseng, beta-sitosterol from Aloe vera, calycosin from Radix Astragali, and extracts from Hippophae rhamnoides L. and Angelica sinensis.

Nucleotide receptors as targets in the pharmacological enhancement of dermal wound healing.

With a growing interest of the involvement of extracellular nucleotides in both normal physiology and pathology, it has become evident that P2 receptor agonists and antagonists may have therapeutic potential. The P2Y2 receptor agonists (diquafosol tetrasodium and denufosol tetrasodium) are in the phase 3 of clinical trials for dry eye and cystic fibrosis, respectively. The thienopyridine derivatives clopidogrel and ticlopidine (antagonists of the platelet P2Y12 receptor) have been used in cardiovascular medicine for nearly a decade. Purines and pyrimidines may be of therapeutic potential also in wound healing since ATP and UTP have been shown to have many hallmarks of wound healing factors. Recent studies have demonstrated that extracellular nucleotides take part in all phases of wound repair: hemostasis, inflammation, tissue formation, and tissue remodeling. This review is focused on the potent purines and pyrimidines which regulate many physiological processes important for wound healing.