15 research outputs found
Quantitative Analysis of Histone Demethylase Probes Using Fluorescence Polarization
We
previously reported methylstat as a selective inhibitor of jumonji
C domain-containing histone demethylases (JHDMs). Herein, we describe
the synthesis of a fluorescent analogue of methylstat and its application
as a tracer in fluorescence polarization assays. Using this format,
we have evaluated the binding affinities of several known JHDM probes,
as well as the native cofactor and substrate of JHDM1A. This fluorophore
allowed a highly robust and miniaturized competition assay sufficient
for high-throughput screening
Recommended from our members
A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load
<div><p>Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen <i>Salmonella enterica (Salmonella)</i> to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAβN (Phe-Arg β-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against <i>Salmonella</i> within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in <i>Salmonella</i> and bound the AcrB subunit of the AcrAB-TolC efflux system with K<sub>D</sub>s in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.</p></div
EPMs synergize with antimicrobial peptides.
<p><i>Salmonella</i> was grown in M9-based defined media in the presence of <b>(A-D</b>) polymyxin B (5 μg/ml; 1/8 MIC) or <b>(E-H)</b> LL37 (5 μg/ml; 1/8 MIC) and EPMs (PAβN, 500 μM; EPMs, 25 μM). Mean and SD of triplicate samples from one representative experiment of three independent biological replicates. DMSO, polymyxin B and LL37 curves repeat across graphs.</p
EPM35 and EPM43 block efflux of Nile red from ESKAPE MDR clinical isolates.
<p>(A-D) Defined strains obtained from BEI resources were examined for Nile red retention after glucose addition in the presence of the indicated compound. Data for each sample were normalized to the initial fluorescence (100%). Dose response curves at seven minutes after glucose addition. Mean of three biological replicates performed in duplicate. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; ****<i>p</i> < 0.0001 compared to DMSO + glucose by one-way ANOVA and Dunnett’s multiple comparison post-test.</p
EPMs block efflux of ethidium bromide and Nile red.
<p><i>Salmonella</i> were incubated with either ethidium bromide or Nile red without glucose, treated with compound, exposed to glucose, and then monitored for fluorescence. Data for each sample were normalized to the initial fluorescence (100%). Dose response curves are (A) ethidium bromide at 28 minutes after glucose addition, (B) Nile red at 7 minutes after glucose addition, and (C) Nile red after washout of compound 7 minutes after glucose addition. Mean of at least two biological replicates performed in duplicate. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; ****<i>p</i> < 0.0001 compared to DMSO + glucose by one-way ANOVA and Dunnett’s multiple comparison post-test.</p
EPMs enhance activity of erythromycin and ciprofloxacin against <i>Salmonella</i> in macrophages.
<p>RAW 264.7 macrophages were infected with <i>Salmonella</i> and treated with a dose range of (A) erythromycin or (B) ciprofloxacin and with DMSO or the indicated concentration of an EPM. At 18 hours post infection samples were processed for fluorescence microscopy as described. Data were normalized to treatment with DMSO and antibiotic vehicle (100%). Key: black, DMSO; red, EPM30; green, EPM35; blue, EPM43; gray, calculated additivity of the antibiotic and the corresponding EPM using the formula (100 –([percent inhibition EPM] + [percent inhibition antibiotic])), where percent inhibition is calculated as 100 –[percent of DMSO]. Data are mean + SEM of three biological replicates. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; **** <i>p</i> < 0.0001 of EPM treatment versus calculated additivity by one-way ANOVA with Sidak’s multiple comparison test.</p
Screening platform (SAFIRE) used to identify EPMs.
<p>(A) Schematic of screening methodology. (B) Representative micrographs of infected macrophages from DMSO-treated wells. Upper left is a field with 522 macrophages; remaining images are the indicated channels zoomed in on the boxed region. Scale bars are 50 μm. (C) Representative micrographs of infected macrophages treated with rifampicin [2 μg/mL] or of uninfected macrophages treated with DMSO. (D) Distribution of B-scores and <i>p</i>-values for 14,400 compounds from the Maybridge HitFinder<sup>TM</sup> v11 library. The locations of the three hit compounds (EPM30, EPM35 and EPM43) and of chloramphenicol (Cm), which was identified from the library, are shown.</p
The three hit compounds decrease bacterial load of <i>Salmonella</i> in mammalian cells.
<p>(A-C) Monitoring of bacterial load by GFP (SAFIRE) or CFU in RAW264.7 cells. (A) Representative micrographs of cells in 96-well plates infected with <i>sifB</i>::<i>GFP Salmonella</i>. Two hours after infection cells were treated with the indicated compound [25 μM] for 16 hours. (B) Dose response curve for SAFIRE and (C) CFU; keys includes IC<sub>50</sub> values. (D-E) Monitoring of bacterial load by GFP (SAFIRE) in HeLa cells infected with <i>Salmonella</i> expressing <i>rpsM</i>::<i>GFP</i> and treated with the indicated compound [25 μM]. (F) Monitoring of bacterial load by CFU in BMDMs treated with compounds [25 μM]. Mean and SEM from three independent biological replicates. The nonlinear curve fitting (B, C) is constrained using uninfected cells as the minimum and DMSO-treated cells as the maximum. (E, F) * <i>p</i> < 0.05; ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 compared to DMSO by one-way ANOVA with Dunnett’s post-test.</p
EPMs do not disrupt bacterial inner or outer membranes.
<p>(A,B) <i>Salmonella</i> treated with DMSO or EPMs [100 μM] but not CCCP [1 mM] acquire TMRM staining within 30 minutes. (A) Representative data from one of three independent experiments. (B) Median fluorescence intensity from three experiments normalized to unstained control (0). (C) Disk diffusion assays; the radius of the zone of growth inhibition after 16 hours of exposure to compound across a dose range. Black lines, semilog fit for the combined antibiotic data; gray lines, semilog fit for CCCP and PAβN; dotted lines, limit of detection (disk radius). Average of two measurements from each image captured from one experiment representative of two independent experiments. (D,E) Nitrocefin access to the periplasm as monitored by nitrocefin [100 μM] hydrolysis in the presence of the indicated concentrations of compounds. (D) Absorbance 486 nm of <i>bla+ Salmonella</i> normalized to <i>bla- Salmonella</i>. Data is representative of at least two independent biological replicates. (E) Slope of the linear region of the A<sub>486</sub> plot from at least three experiments. Data is normalized to A<sub>486</sub>/minute. * <i>p</i> < 0.05, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001 by one-way ANOVA with Dunnett’s post-test.</p