Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics

A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, ObgGC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z’ value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between ObgGC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an ObgGC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of ObgGC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteria

Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics

A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, Obg[subscript]GC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z’ value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between Obg[subscript]GC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an Obg[subscript]GC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of Obg[subscript]GC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteri

The impact of aminated surface ligands and silica shells on the stability, uptake, and toxicity of engineered silver nanoparticles

Inherent nanomaterial characteristics, composition, surface chemistry, and primary particle size, are known to impact particle stability, uptake, and toxicity. Nanocomposites challenge our ability to predict nanoparticle reactivity in biological systems if they are composed of materials with contrasting relative toxicities. We hypothesized that toxicity would be dominated by the nanoparticle surface (shell vs core), and that modulating the surface ligands would have a direct impact on uptake. We exposed developing zebrafish (Danio rerio) to a series of ~70 nm amine-terminated silver nanoparticles with silica shells (AgSi NPs) to investigate the relative influence of surface amination, composition, and size on toxicity. Like-sized aminated AgSi and Si NPs were more toxic than paired hydroxyl-terminated nanoparticles; however, both AgSi NPs were more toxic than the Si NPs, indicating a significant contribution of the silver core to the toxicity. Incremental increases in surface amination did not linearly increase uptake and toxicity, but did have a marked impact on dispersion stability. Mass-based exposure metrics initially supported the hypothesis that smaller nanoparticles (20 nm) would be more toxic than larger particles (70 nm). However, surface area-based metrics revealed that toxicity was independent of size. Our studies suggest that nanoparticle surfaces play a critical role in the uptake and toxicity of AgSi NPs, while the impact of size may be a function of the exposure metric used. Overall, uptake and toxicity can be dramatically altered by small changes in surface functionalization or exposure media. Only after understanding the magnitude of these changes, can we begin to understand the biologically available dose following nanoparticle exposure.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Springer. The published article can be found at: http://link.springer.com/journal/11051.Keywords: Dispersion, Zebrafish, Nanomaterials, Environmental and health effects, Surface chemistry, Silica shel

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen–responsive reproductive and biomarker genes

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen–responsive genes in this model population. First, a dose–response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17β–estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER βa, ER βb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17β–estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi–chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10 ng/g 17β–estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17β–estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen–responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.Keywords: Killifish, Vitellogenin, Choriogenin, Estrogen, Biomarkers, Endocrine disruptionKeywords: Killifish, Vitellogenin, Choriogenin, Estrogen, Biomarkers, Endocrine disruptio

BonventreJosephineEMTImpactAminatedSurface_SupportingInformation.zip

Inherent nanomaterial characteristics, composition, surface chemistry, and primary particle size, are known to impact particle stability, uptake, and toxicity. Nanocomposites challenge our ability to predict nanoparticle reactivity in biological systems if they are composed of materials with contrasting relative toxicities. We hypothesized that toxicity would be dominated by the nanoparticle surface (shell vs core), and that modulating the surface ligands would have a direct impact on uptake. We exposed developing zebrafish (Danio rerio) to a series of ~70 nm amine-terminated silver nanoparticles with silica shells (AgSi NPs) to investigate the relative influence of surface amination, composition, and size on toxicity. Like-sized aminated AgSi and Si NPs were more toxic than paired hydroxyl-terminated nanoparticles; however, both AgSi NPs were more toxic than the Si NPs, indicating a significant contribution of the silver core to the toxicity. Incremental increases in surface amination did not linearly increase uptake and toxicity, but did have a marked impact on dispersion stability. Mass-based exposure metrics initially supported the hypothesis that smaller nanoparticles (20 nm) would be more toxic than larger particles (70 nm). However, surface area-based metrics revealed that toxicity was independent of size. Our studies suggest that nanoparticle surfaces play a critical role in the uptake and toxicity of AgSi NPs, while the impact of size may be a function of the exposure metric used. Overall, uptake and toxicity can be dramatically altered by small changes in surface functionalization or exposure media. Only after understanding the magnitude of these changes, can we begin to understand the biologically available dose following nanoparticle exposure.Keywords: Silica shell, Environmental and health effects, Zebrafish, Surface chemistry, Dispersion, Nanomaterial

Assay optimization.

<p>The concentrations of Obg_GC and GTP were selected to achieve an optimal signal window for the HTS assay using as a read-out colorimetric free phosphate quantitation with BIOMOLGreen. <b>(A)</b> Various concentrations of Obg_GC (1–10 μM) were incubated with 125 μM GTP for 6 h at 37°C, followed by measurement of free phosphate amounts. <b>(B)</b> Obg_GC (5 μM) was incubated with increasing concentrations of GTP (125–1000 μM) for 6 h at 37°C and the free phosphate present in each reaction was quantitated. <b>(C)</b> Time course with 5 μM Obg_GC and 250 μM GTP assayed at 0, 1, 6, 18, 24 and 36 h demonstrated that the Obg-dependent GTP hydrolysis continued to increase until approximately 18 h. <b>(D)</b> Obg_GC (5 μM) was incubated with GTP (250 μM) in the presence or absence of Mg²⁺ for 6 or 18 h at either 37°C or room temperature (RT). The signal windows for the three incubation conditions were 2.33, 4.25, and 2.76, respectively, indicating that 18 h incubation time at 37°C was the most optimal. There was no significant difference between free phosphate amounts detected in reactions lacking Mg²⁺ (red bars) and that of buffer alone (background, white bars). Asterisk denotes significant difference between complete reaction (blue bars) and reaction mixture lacking Mg²⁺ (ANOVA, P < 0.0001).</p

Biochemical analysis of Obg isolated from <i>K</i>. <i>pneumoniae</i> and MRSA.

<p>To evaluate the potential of broad-spectrum activity of lead compounds identified using Obg_GC as a target, Obg_KP and Obg_MRSA were purified and GTP hydrolysis as well as GTP and GDP binding were examined using the colorimetric (A) and fluorescence-based activity assays using mant-nucleotides (B, C and D). <b>(A)</b> The free phosphate detection assay was conducted with 5 μM protein and 250 μM GTP and the reactions were incubated for 18 h at 37°C followed by the addition of BIOMOL Green reagents and absorbance measurements. Similar concentrations of phosphate were detected with both Obg_KP and Obg_MRSA (not significantly different, t-test, P = 0.3109). Both proteins required the presence of Mg²⁺ to hydrolyze GTP (significantly different, respectively, T-TEST P< 0.0001). The data shows averages, with corresponding SEM, of six biological replicates. <b>(B)</b> Binding of mant-GTP (orange bars) and mant-GDP (yellow bars) to Obg_KP and Obg_MRSA was examined in the presence and absence of Mg²⁺, as indicated below the graph. Obg_KP binding of mant-GTP and mant-GDP increased the RFU ~1.8- and 1.3-fold, respectively. Whereas ~1.4- and 1.1-fold increase in RFU was observed for the corresponding mant-nucleotides upon binding by Obg_MRSA. There was a significant difference in ability of Obg_KP and Obg_MRSA to bind mant-GTP (t-test, P <0.001). The reliance on Mg²⁺ for both proteins was demonstrated by the absence of an increase in RFU in reaction buffer deficient in Mg²⁺ (Significantly different, respectively, t-test, P< 0.0001). The data shows averages with corresponding SEM of five experiments performed on separate occasions. Hydrolysis of mant-GTP by Obg_KP <b>(C)</b> and Obg_MRSA <b>(D)</b> was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes over 3 h. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black. The first-order rate constant, k_<i>h</i>, of 4.64 × 10⁻⁴s⁻¹ and 6.36 × 10⁻⁴s⁻¹, and half-life (T_1/2) of 24.88 min and 18.17 min, were calculated for Obg_KP and Obg_MRSA, respectively.</p

Statistical analysis of assay performance.

<p>Statistical analysis of assay performance.</p

Assessments of solvent tolerance and chelator challenge.

<p>Potential complications of screening conditions were addressed by challenging Obg_GC with various concentrations of DMSO <b>(A)</b> or multiple known chelators <b>(B)</b> followed by measurement of GTPase Obg_GC activity using free phosphate quantitation with BIOMOL Green. Protein activity was similar in the presence of 0–5% DMSO and in the presence of three common chelators EDTA, EGTA, and NTA tested 40 μM final concentration. CA significantly decreased the amount of phosphate detected, but it also decreased the background phosphate in the reaction lacking Obg_GC (not shown), and was therefore altering the absorbance of the reagent, not acting as a chelator. Asterisk denotes significant difference (ANOVA, P < 0.0001).</p