43 research outputs found
High-Throughput Screening To Identify Potent and Specific Inhibitors of Microbial Sulfate Reduction
The selective perturbation of complex
microbial ecosystems to predictably
influence outcomes in engineered and industrial environments remains
a grand challenge for geomicrobiology. In some industrial ecosystems,
such as oil reservoirs, sulfate reducing microorganisms (SRM) produce
hydrogen sulfide which is toxic, explosive, and corrosive. Despite
the economic cost of sulfidogenesis, there has been minimal exploration
of the chemical space of possible inhibitory compounds, and very little
work has quantitatively assessed the selectivity of putative souring
treatments. We have developed a high-throughput screening strategy
to identify potent and selective inhibitors of SRM, quantitatively
ranked the selectivity and potency of hundreds of compounds and identified
previously unrecognized SRM selective inhibitors and synergistic interactions
between inhibitors. Zinc pyrithione is the most potent inhibitor of
sulfidogenesis that we identified, and is several orders of magnitude
more potent than commonly used industrial biocides. Both zinc and
copper pyrithione are also moderately selective against SRM. The high-throughput
(HT) approach we present can be readily adapted to target SRM in diverse
environments and similar strategies could be used to quantify the
potency and selectivity of inhibitors of a variety of microbial metabolisms.
Our findings and approach are relevant to efforts to engineer environmental
ecosystems and also to understand the role of natural gradients in
shaping microbial niche space
MOESM3 of Obstacles to successful treatment of hepatitis C in uninsured patients from a minority population
Additional file 3: Table S2. Univariable and multivariable logistic regression for association with being tested for SVR
Treatment of infected THP-1 with DMSO and amphotericin B.
<p>A. Number of infected THP-1 counted per well treated or not with 1% DMSO or 2 µM amphotericin B. B. Number of parasites counted per well divided by the number of host nuclei per field. C. Dose response curve for amphotericin B plotting the percentage of parasite growth inhibition. Values are mean from at least 3 independent experiments.</p
MOESM2 of Obstacles to successful treatment of hepatitis C in uninsured patients from a minority population
Additional file 2: Table S1. Univariable and multivariable logistic regression for association with treatment completion
MOESM1 of Obstacles to successful treatment of hepatitis C in uninsured patients from a minority population
Additional file 1: Figure S1. Attainment of specific milestones in the HCV treatment cascade of cirrhotic and non-cirrhotic patients. A. In patients who initiated treatment (N = 216). B. In patients who completed treatment (N = 189). C. In patients who obtained a 12-week post treatment viral load (N = 177). †Completed treatment per guidelines (based on patient report)
Number of hits identified with the intracellular amastigote and the promastigote primary screens.
<p>White bar: number of compounds identified in both screens. Light grey and black bars: number of compounds specifically active against promastigotes and intracellular amastigotes respectively. Hatched bar: number of compounds active against the promastigote stage but determined as toxic against THP-1 host cell in the intracellular amastigote screen.</p
Infection of THP-1 with <i>L. donovani</i>: detection, segmentation and growth of host cell and parasite.
<p>A–D. Detection and segmentation of THP-1 host cell and <i>L. donovani</i> intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with <i>L. donovani</i> and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and <i>L. donovani</i> were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).</p
Structure and activity of naloxonazine and naloxone.
<p>Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.</p
Diverse Inhibitor Chemotypes Targeting <em>Trypanosoma cruzi</em> CYP51
<div><h3>Background</h3><p>Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.</p> <h3>Methodology/Principal Findings</h3><p>The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon <em>Trypanosoma cruzi</em>, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the <em>T. cruzi</em> CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K<em><sub>D</sub></em> values, while cross-validation against <em>T. cruzi</em>-infected skeletal myoblast cells yielded 57 active hits with EC<sub>50</sub> <10 µM. Two pools of hits partially overlapped. The top hit inhibited <em>T. cruzi</em> with EC<sub>50</sub> of 17 nM and was trypanocidal at 40 nM.</p> <h3>Conclusions/Significance</h3><p>The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against <em>T. cruzi</em>.</p> </div
Flowchart diagram of screening and validation steps.
<p>Flowchart diagram of screening and validation steps.</p