Identification and Characterization of Inhibitors of the Aminoglycoside Resistance Acetyltransferase Eis from Mycobacterium tuberculosis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89566/1/73_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/89566/2/cmdc_201100332_sm_miscellaneous_information.pd

Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen.

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well regulated, persistent neutrophils may cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real-time. Finally, our studies reveal that clinically available ‘cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings may have implications for the therapeutic use of benzopyrones in inflammatory disease

Development of small-molecule fluorescent probes targeting neutrophils via N-formyl peptide receptors

N-Formyl peptide receptors (FPRs) are membrane receptors that are abundantly expressed in innate immune cells, including neutrophils and platelets, demonstrating potential new targets for immune system regulation and the treatment of inflammatory conditions. We report here the development and bio-physical validation of new FPR imaging agents as effective tools to track FPR distribution, localisation and functions, ultimately helping to establish FPR exact roles and functions in pathological and physiological conditions. The new series of probes feature a small molecule-based FPR address system conjugated to suitable fluorophores, resulting in highly specific FPR agents, including a partial agonist endowed with high affinity (i.e. low/sub-nanomolar potency) on FPR-transfected cells and human neutrophils. Preliminary imaging studies via multiphoton microscopy demonstrate that the probes enable the visualisation of FPRs in live cells, thus representing valid bio-imaging tools for the analysis of FPR-mediated signalling, such as the activation of neutrophils in inflammatory events.</p

Polysaccharides Isolated from Açaí Fruit Induce Innate Immune Responses

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease

Modulation of Human Neutrophil Responses by the Essential Oils from Ferula akitschkensis and Their Constituents

WOS: 000384518200014PubMed ID: 27586050Essential oils were obtained by hydrodistillation of the umbels+seeds and stems of Ferula akitschkensis (FAEO(u/s) and FAEO(stm), respectively) and analyzed by gas chromatography and gas chromatography-mass spectrometry. Fifty-two compounds were identified in FASO(u/s); the primary components were sabinene, alpha-pinene, beta-pinene, terpinen-4-ol, eremophilene, and 2-himachalen-7-ol, whereas the primary components of FAEOstm were myristicin and geranylacetone. FAEO(u/s), beta-pinene, sabinene, gamma-terpinene, geranylacetone, isobornyl acetate, and (E)-2-nonenal stimulated [Ca2+](i) mobilization in human neutrophils, with the most potent being geranylacetone (EC50 = 7.6 +/- 1.9 mu M) and isobornyl acetate 6.4 +/- 1.7 (EC50 = 7.6 +/- 1.9 mu M). In addition, treatment of neutrophils with beta-pinene, sabinene, gamma-terpinene, geranylacetone, and isobornyl acetate desensitized the cells to N-formyl-Met-Leu-Phe (fMLF)- and interleukin-8 (IL-8)-induced [Ca2+](i) flux and inhibited fMLF-induced chemotaxis. The effects of beta-pinene, sabinene, gamma-terpinene, geranylacetone, and isobornyl acetate on neutrophil [Ca2+](i) flux were inhibited by transient receptor potential (TRP) channel blockers. Furthermore, the most potent compound, geranylacetone, activated Ca2+ influx in TRPV1-transfected HEK293 cells. In contrast, myristicin inhibited neutrophil [Ca2+](i) flux stimulated by fMLF and IL-8 and inhibited capsaicin-induced Ca2+ influx in TRPV1-transfected HEK293 cells. These findings, as well as pharmacophore modeling of TRP agonists, suggest that geranylacetone is a TRPV1 agonist, whereas myristicin is a TRPV1 antagonist. Thus, at least part of the medicinal properties of Ferula essential oils may be due to modulatory effects on TRP channels.National Institutes of Health IDeA Program COBRE Grant [GM110732]; Ministry of Education and Science, Kazakhstan [0504/GF3, 2117/GF4]; USDA National Institute of Food and Agriculture Hatch project [1009546]; Montana University System Research Initiative [51040-MUSRI2015-03]; Montana State University Agricultural Experiment StationThis research was supported in part by National Institutes of Health IDeA Program COBRE Grant GM110732; Grants 0504/GF3 and 2117/GF4 from the Ministry of Education and Science, Kazakhstan; a USDA National Institute of Food and Agriculture Hatch project 1009546; Montana University System Research Initiative 51040-MUSRI2015-03; and the Montana State University Agricultural Experiment Station

Inhibition of Human Neutrophil Responses by the Essential Oil of Artemisia kotuchovii and Its Constituents

WOS: 000355383700011PubMed ID: 25959257Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEO(f+l) and AKEO(stm), respectively) and analyzed by gas chromatography (GC) and gas chromatography,mass spectrometry (GC-MS). The primary components of the oils were estragole, (E)- and (Z)-beta-ocimenes, methyleugenol, limonene, spathulenol, beta-pinene, myrcene, and (E)-methyl cinnamate. Seventy-four constituents were present at concentrations from 0.1 to 1.0%, and 34 compounds were identified in trace (96% of the AKEO(stm), composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). One component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca2+ flux, fMLF-indticed chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca2+ flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration and ROS production.National Institutes of Health IDeA Program COBRE Grant [GM110732]; USDA National Institute of Food and Agriculture Hatch project; Montana State University Agricultural Experiment Station; Ministry of Education and Science, Kazakhstan [0504/GF3, 2117/GF4]This research was supported in part by National Institutes of Health IDeA Program COBRE Grant GM110732; Grants 0504/GF3 and 2117/GF4 from The Ministry of Education and Science, Kazakhstan; a USDA National Institute of Food and Agriculture Hatch project; and the Montana State University Agricultural Experiment Station