Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases

Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease.

Inflammatory bowel disease (IBD) continues to increase in prevalence in industrialized countries. Major complications of IBD include formation of fibrotic strictures, fistulas, reduced absorptive function, cancer risk, and the need for surgery. In other chronic gastrointestinal disease models, stiffness has been shown to precede fibrosis; therefore, stiffness may be a reasonable indicator of progression toward stricture formation in IBD patients. Herein, we seek to quantify tissue stiffness and characterize fibrosis in patients with IBD and to compare mechanical properties of unaffected human tissue to common animal species used for IBD studies. Inflamed and unaffected tissue from IBD patients and unaffected tissue from mice, pigs, and cows were indented using a custom device to determine the effective stiffness. Histology was performed on matched tissues, and total RNA was isolated from IBD tissue samples and used for gene expression analysis of pro-fibrotic genes. We observed an increase in the effective stiffness (steady-state modulus, SSM) (p < 0.0001) and increased expression of the collagen type I gene (COL1A1, p = 0.01) in inflamed tissue compared to unaffected areas in our IBD patient cohort. We also found that increased staining of collagen fibers in submucosa positively correlated with SSM (p = 0.093). We determined that unaffected animal bowel stiffness is significantly greater than similar human tissues, suggesting additional limitations on animal models for translational investigations regarding stiffness-related hypotheses. Taken together, our data support development of tools for evaluation of bowel stiffness in IBD patients for prognostic applications that may enable more accurate prediction of those who will develop fibrosis and more precise prescription of aggressive therapies

Comparison of the steady-state modulus (SSM) and effective total modulus of human tissue and control animal tissue.

<p>Inflamed human tissue is displayed in orange while unaffected human and normal animal tissue is displayed in blue. (a) The SSM is representative of the steady-state behavior of a viscoelastic material after stress relaxation (Eqs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e002" target="_blank">2</a>). (b) The effective total modulus is determined from using the Winkler contact model which assumes that the tissue is a foundation of purely elastic springs (Eq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e003" target="_blank">3</a>). Group means are displayed as a solid black line within each group. Each dot represents one indentation. SI = small intestine, LI = large intestine. * p < 0.0001, ** p = 0.0004, ‡ p<0.0001, ‡‡ p = 0.0043. Note: Scales of y-axes are different for each graph as effective total modulus yields higher values than SSM.</p

The steady-state modulus of human colon and ileum vary with inflammation state.

<p>(A) The difference in stiffness between colon and ileum was not significant (p = 0.30, n = 44 colon measurements on 4 samples from 3 patients and n = 33 ileum measurements on 9 samples from 9 patients). (B) Inflamed regions are stiffer than unaffected regions (p < 0.0001, n = 43 inflamed indentations from 7 patients and n = 34 unaffected indentations from 6 patients). Following this trend, inflamed colonic tissue (n = 31 indentations from 3 patients) is stiffer than unaffected colon regions (n = 13 from 1 patient) and the inflamed ileum (n = 12 from 4 patients) is stiffer than unaffected ileal tissue (n = 21 from 5 patients). Mean stiffness values are represented as a solid black bar.</p

Multi-scale indentation system characterizes mechanical properties of soft tissues.

<p>(A) Custom indentation device with sensor positioned with the cantilever in position to test inflamed ileum. (B) Bovine small bowel positioned under the sensor probe with the mucosa exposed. Hydration was maintained by pooling small volumes of saline solution on and around samples (not seen here to improve visualization of tissue).</p

Tissues with time-dependent mechanical properties reach a quasi-steady-state after stress relaxation during indentation.

<p>Normal force (top, green line) as a function of relaxation time is used in computing the transient modulus (bottom, blue line) based on a modified Hertz contact model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e001" target="_blank">Equation 1</a>). The Steady-State Modulus (SSM) seen at the end of relaxation is reported throughout this work since SSM reduces strain-rate dependencies in determining the properties of a viscoelastic material.</p

mRNA expression of Col1A1, MMP-1, and CDH-1 is changed in unaffected and inflamed areas of the intestine.

<p>mRNA expression profiles of Col1A1 (A), MMP-1 (B), and CDH1 (C) in inflamed and unaffected areas of IBD patients. N = 9 for each group and is represented as the average of 3 separate experiments. Collagen type I is significantly upregulated in the inflamed areas (*p = 0.0110) along with MMP-1 expression upregulation in inflamed areas (***p = 0.0032). The expression of CDH1, E-cadherin, is downregulated in inflamed areas (*p = 0.0288). Gene expression that resulted in no significant change is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.s001" target="_blank">S1 Fig</a>.</p

Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease - Fig 8

<p><b>Representative H&E and Masson’s Trichrome of the cow small bowel (A,B), pig small bowel (C,D), pig colon (E,F), and mouse colon (G,H).</b> The mouse colon has a very distinct muscle layer highlighted by the black arrow (G), while the cow small bowel has a very large area of collagen deposition (B, black star) that is absent in the pig small bowel and colon (black stars, D and F). Scale bar is 100 μm.</p

Fibrotic markers are less prevalent in representative staining of the unaffected regions of bowel than inflamed regions.

<p>Adjacent regions of Ileum (A-D) and colon (E-H) have been labeled with H&E (A,C,E,G) and Trichrome (B,D,F,H) as indicated. Black arrows highlight collagen clustering while black stars illustrate disrupted mucosal layer in inflamed tissue compared to unaffected tissue segments. Scale bar is 100 μm.</p