Pharmacological inhibition of TRPV2 attenuates phagocytosis and lipopolysaccharide-induced migration of primary macrophages.

BACKGROUND AND PURPOSE: In macrophages, transient receptor potential vanilloid 2 (TRPV2) channel contributes to various cellular processes such as cytokine production, differentiation, phagocytosis and migration. Due to a lack of selective pharmacological tools, its function in immunological processes is not well understood and the identification of novel and selective TRPV2 modulators is highly desirable. EXPERIMENTAL APPROACH: Novel and selective TRPV2 modulators were identified by screening a compound library using Ca2+ influx assays with human embryonic kidney 293 (HEK293) cells heterologously expressing rat TRPV2. Hits were further characterized and validated with Ca2+ influx and electrophysiological assays. Phagocytosis and migration of macrophages were analysed and the contribution of TRPV2 to the generation of Ca2+ microdomains was studied by total internal reflection fluorescence microscopy (TIRFM). KEY RESULTS: The compound IV2-1, a dithiolane derivative (1,3-dithiolan-2-ylidene)-4-methyl-5-phenylpentan-2-one), is a potent inhibitor of heterologously expressed TRPV2 channels (IC50 = 6.3 ± 0.7 µM) but does not modify TRPV1, TRPV3 or TRPV4 channels. IV2-1 also inhibits TRPV2-mediated Ca2+ influx in macrophages. IV2-1 inhibits macrophage phagocytosis along with valdecoxib and after siRNA-mediated knockdown. Moreover, TRPV2 inhibition inhibits lipopolysaccharide-induced migration of macrophages whereas TRPV2 activation promotes migration. After activation, TRPV2 shapes Ca2+ microdomains predominantly at the margin of macrophages, which are important cellular regions to promote phagocytosis and migration. CONCLUSIONS AND IMPLICATIONS: IV2-1 is a novel TRPV2-selective blocker and underline the role of TRPV2 in macrophage-mediated phagocytosis and migration. Furthermore, we provide evidence that TRPV2 activation generates Ca2+ microdomains, which may be involved in phagocytosis and migration of macrophages.

Droplet Microarray as a Powerful Platform for Seeking New Antibiotics Against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria is a severe threat to public health. Therefore, it is urgent to establish effective screening systems for identifying novel antibacterial compounds. In this study, a highly miniaturized droplet microarray (DMA) based high-throughput screening system is established to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA). The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders. Due to the differences in the surface wettability between the spots and the borders, arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs can be generated for screening applications. A simple colorimetric viability readout utilizing a conventional photo scanner is developed for fast single-step detection of the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system enables the identification of potential antibiotics from novel synthetic compound libraries, offering opportunities for development of new treatments against multidrug-resistant bacteria.

Platinum Cyclooctadiene Complexes with Activity against Gram-positive Bacteria.

Antimicrobial resistance is a looming health crisis, and it is becoming increasingly clear that organic chemistry alone is not sufficient to continue to provide the world with novel and effective antibiotics. Recently there has been an increased number of reports describing promising antimicrobial properties of metal-containing compounds. Platinum complexes are well known in the field of inorganic medicinal chemistry for their tremendous success as anticancer agents. Here we report on the promising antibacterial properties of platinum cyclooctadiene (COD) complexes. Amongst the 15 compounds studied, the simplest compounds Pt(COD)X2 (X=Cl, I, Pt1 and Pt2) showed excellent activity against a panel of Gram-positive bacteria including vancomycin and methicillin resistant Staphylococcus aureus. Additionally, the lead compounds show no toxicity against mammalian cells or haemolytic properties at the highest tested concentrations, indicating that the observed activity is specific against bacteria. Finally, these compounds showed no toxicity against Galleria mellonella at the highest measured concentrations. However, preliminary efficacy studies in the same animal model found no decrease in bacterial load upon treatment with Pt1 and Pt2. Serum exchange studies suggest that these compounds exhibit high serum binding which reduces their bioavailability in vivo, mandating alternative administration routes such as e. g. topical application.

Effects of a Novel GPR55 Antagonist on the Arachidonic Acid Cascade in LPS-Activated Primary Microglial Cells.

Neuroinflammation is a crucial process to maintain homeostasis in the central nervous system (CNS). However, chronic neuroinflammation is detrimental, and it is described in the pathogenesis of CNS disorders, including Alzheimer's disease (AD) and depression. This process is characterized by the activation of immune cells, mainly microglia. The role of the orphan G-protein-coupled receptor 55 (GPR55) in inflammation has been reported in different models. However, its role in neuroinflammation in respect to the arachidonic acid (AA) cascade in activated microglia is still lacking of comprehension. Therefore, we synthesized a novel GPR55 antagonist (KIT 10, 0.1-25 µM) and tested its effects on the AA cascade in lipopolysaccharide (LPS, 10 ng / mL)-treated primary rat microglia using Western blot and EIAs. We show here that KIT 10 potently prevented the release of prostaglandin E2 (PGE2), reduced microsomal PGE2 synthase (mPGES-1) and cyclooxygenase-2 (COX-2) synthesis, and inhibited the phosphorylation of Ikappa B-alpha (IκB-α), a crucial upstream step of the inflammation-related nuclear factor-kappaB (NF-κB) signaling pathway. However, no effects were observed on COX-1 and -2 activities and mitogen-activated kinases (MAPK). In summary, the novel GPR55 receptor antagonist KIT 10 reduces neuroinflammatory parameters in microglia by inhibiting the COX-2/PGE2 pathway. Further experiments are necessary to better elucidate its effects and mechanisms. Nevertheless, the modulation of inflammation by GPR55 might be a new therapeutic option to treat CNS disorders with a neuroinflammatory background such as AD or depression.

Naturally Occurring Cardenolides Affecting Schistosoma mansoni.

Schistosomiasis is a neglected tropical disease of considerable public health burden. We recently discovered a micromolar activity of several cardenolides against newly transformed schistosomula (NTS) of the parasitic flatworm Schistosoma mansoni in a small compound screen including different substance classes of both natural products as well as synthetic molecules. In further experiments, a focused library of naturally occurring and synthetic steroids was explored against NTS and adult S. mansoni, revealing seven cardenolides with comparable activities as known anthelminthics such as praziquantel. Of these, gomphoside monoacetate and uscharin showed suitable therapeutic indices. In a first in vivo study, at a dose of 10 mg/kg, only minor activity in mice harboring a chronic S. mansoni infection could be shown, which will be further investigated by structure-activity relationship studies as well as pharmacodynamic and pharmacokinetic approaches.